In 1540, James V had toured the Hebrides, forcing the clan chiefs to accompany him. There followed a period of peace, but the clans were soon at loggerheads with one another again. During James VI's reign, the citizens of the Hebrides were portrayed as lawless barbarians rather than being the cradle of Scottish Christianity and nationhood. Official documents describe the peoples of the Highlands as "void of the knawledge and feir of God" who were prone to "all kynd of barbarous and bestile cruelteis". The Gaelic language, spoken fluently by James IV and probably by James V, became known in the time of James VI as "Erse" or Irish, implying that it was foreign in nature. The Scottish Parliament decided that Gaelic had become a principal cause of the Highlanders' shortcomings and sought to abolish it.

James's visit to Denmark, a country familiar with witch-hunts, sparked an interest in the study of witchcraft, which he considered a branch of theology. He attended the North Berwick witch trials, the first major persecution of witches in Scotland under the Witchcraft Act 1563. Several people were convicted of using witchcraft to send storms against James's ship, most notably Agnes Sampson.

James was born on 19 June 1566 at Edinburgh Castle, and as the eldest son and heir apparent of the monarch automatically became Duke of Rothesay and Prince and Great Steward of Scotland. He was baptised "Charles James" or "James Charles" on 17 December 1566 in a Catholic ceremony held at Stirling Castle. His godparents were Charles IX of France (represented by John, Count of Brienne), Elizabeth I of England (represented by the Earl of Bedford), and Emmanuel Philibert, Duke of Savoy (represented by ambassador Philibert du Croc). Mary refused to let the Archbishop of St Andrews, whom she referred to as "a pocky priest", spit in the child's mouth, as was then the custom. The subsequent entertainment, devised by Frenchman Bastian Pagez, featured men dressed as satyrs and sporting tails, to which the English guests took offence, thinking the satyrs "done against them".

The care of James was entrusted to the Earl and Countess of Mar, "to be conserved, nursed, and upbrought" in the security of Stirling Castle. James was anointed King of Scots at the age of thirteen months at the Church of the Holy Rude, Stirling, by Adam Bothwell, Bishop of Orkney, on 29 July 1567. The sermon at the coronation was preached by John Knox. In accordance with the religious beliefs of most of the Scottish ruling class, James was brought up as a member of the Protestant Church of Scotland, the Kirk. The Privy Council selected George Buchanan, Peter Young, Adam Erskine (lay abbot of Cambuskenneth), and David Erskine (lay abbot of Dryburgh) as James's preceptors or tutors. As the young king's senior tutor, Buchanan subjected James to regular beatings but also instilled in him a lifelong passion for literature and learning. Buchanan sought to turn James into a God-fearing, Protestant king who accepted the limitations of monarchy, as outlined in his treatise De Jure Regni apud Scotos.

In 1568, Mary escaped from her imprisonment at Loch Leven Castle, leading to several years of sporadic violence. The Earl of Moray defeated Mary's troops at the Battle of Langside, forcing her to flee to England, where she was subsequently kept in confinement by Elizabeth. On 23 January 1570, Moray was assassinated by James Hamilton of Bothwellhaugh. The next regent was James's paternal grandfather Matthew Stewart, 4th Earl of Lennox, who was carried fatally wounded into Stirling Castle a year later after a raid by Mary's supporters. His successor, the Earl of Mar, "took a vehement sickness" and died on 28 October 1572 at Stirling. Mar's illness, wrote James Melville, followed a banquet at Dalkeith Palace given by James Douglas, 4th Earl of Morton.

James was the son of Mary, Queen of Scots, and a great-great-grandson of Henry VII, King of England and Lord of Ireland, positioning him to eventually accede to all three thrones. James succeeded to the Scottish throne at the age of thirteen months, after his mother was compelled to abdicate in his favour. Four different regents governed during his minority, which ended officially in 1578, though he did not gain full control of his government until 1583. In 1603, he succeeded the last Tudor monarch of England and Ireland, Elizabeth I, who died without issue. He continued to reign in all three kingdoms for 22 years, a period known after him as the Jacobean era, until his death in 1625 at the age of 58. After the Union of the Crowns, he based himself in England (the largest of the three realms) from 1603, only returning to Scotland once in 1617, and styled himself "King of Great Britain and Ireland". He was a major advocate of a single parliament for England and Scotland. In his reign, the Plantation of Ulster and British colonisation of the Americas began.

In the 1580s and 1590s, James promoted the literature of his native country. He published his treatise Some Rules and Cautions to be Observed and Eschewed in Scottish Prosody in 1584 at the age of 18. It was both a poetic manual and a description of the poetic tradition in his mother tongue of Scots, applying Renaissance principles. He also made statutory provision to reform and promote the teaching of music, seeing the two in connection. One act of his reign urges the Scottish burghs to reform and support the teaching of music in Sang Sculis.

Morton was elected to Mar's office and proved in many ways the most effective of James's regents, but he made enemies by his rapacity. He fell from favour when Frenchman Esmé Stewart, Sieur d'Aubigny, first cousin of James's father Lord Darnley and Future Earl of Lennox, arrived in Scotland and quickly established himself as the first of James's powerful favourites. Morton was executed on 2 June 1581, belatedly charged with complicity in Darnley's murder. On 8 August, James made Lennox the only duke in Scotland. The king, then fifteen years old, remained under the influence of Lennox for about one more year.

Lennox was a Protestant convert, but he was distrusted by Scottish Calvinists who noticed the physical displays of affection between him and the king and alleged that Lennox "went about to draw the King to carnal lust". In August 1582, in what became known as the Ruthven Raid, the Protestant earls of Gowrie and Angus lured James into Ruthven Castle, imprisoned him, and forced Lennox to leave Scotland. During James's imprisonment (19 September 1582), John Craig, whom the king had personally appointed Royal Chaplain in 1579, rebuked him so sharply from the pulpit for having issued a proclamation so offensive to the clergy "that the king wept".

After James was liberated in June 1583, he assumed increasing control of his kingdom. He pushed through the Black Acts to assert royal authority over the Kirk, and denounced the writings of his former tutor Buchanan. Between 1584 and 1603, he established effective royal government and relative peace among the lords, ably assisted by John Maitland of Thirlestane who led the government until 1592. An eight-man commission known as the Octavians brought some control over the ruinous state of James's finances in 1596, but it drew opposition from vested interests. It was disbanded within a year after a riot in Edinburgh, which was stoked by anti-Catholicism and led the court to withdraw to Linlithgow temporarily.

In 1586, James signed the Treaty of Berwick with England. That and the execution of his mother in 1587, which he denounced as a "preposterous and strange procedure", helped clear the way for his succession south of the border. Queen Elizabeth was unmarried and childless, and James was her most likely successor. Securing the English succession became a cornerstone of his policy. During the Spanish Armada crisis of 1588, he assured Elizabeth of his support as "your natural son and compatriot of your country".

By the late 1590s, his championing of native Scottish tradition was reduced to some extent by the increasing likelihood of his succession to the English throne. william Alexander and other courtier poets started to anglicise their written language, and followed the king to London after 1603. James's role as active literary participant and patron made him a defining figure in many respects for English Renaissance poetry and drama, which reached a pinnacle of achievement in his reign, but his patronage of the high style in the Scottish tradition, which included his ancestor James I of Scotland, became largely sidelined.

In 1597–98, James wrote The True Law of Free Monarchies and Basilikon Doron (Royal Gift), in which he argues a theological basis for monarchy. In the True Law, he sets out the Divine right of kings, explaining that kings are higher beings than other men for Biblical reasons, though "the highest bench is the sliddriest to sit upon". The document proposes an absolutist theory of monarchy, by which a king may impose new laws by royal prerogative but must also pay heed to tradition and to God, who would "stirre up such scourges as pleaseth him, for punishment of wicked kings".

It was against this background that James VI authorised the "Gentleman Adventurers of Fife" to civilise the "most barbarous Isle of Lewis" in 1598. James wrote that the colonists were to act "not by agreement" with the local inhabitants, but "by extirpation of thame". Their landing at Stornoway began well, but the colonists were driven out by local forces commanded by Murdoch and Neil MacLeod. The colonists tried again in 1605 with the same result, although a third attempt in 1607 was more successful. The Statutes of Iona were enacted in 1609, which required clan chiefs to: send their heirs to Lowland Scotland to be educated in English-speaking Protestant schools; provide support for Protestant ministers to Highland parishes; outlaw bards; and regularly report to Edinburgh to answer for their actions. So began a process "specifically aimed at the extirpation of the Gaelic language, the destruction of its traditional culture and the suppression of its bearers."

One last Scottish attempt against the king's person occurred in August 1600, when James was apparently assaulted by Alexander Ruthven, the Earl of Gowrie's younger brother, at Gowrie House, the seat of the Ruthvens. Ruthven was run through by James's page John Ramsay and the Earl of Gowrie was killed in the ensuing fracas; there were few surviving witnesses. Given James's history with the Ruthvens and the fact that he owed them a great deal of money, his account of the circumstances was not universally believed.

Elizabeth I was the last of Henry VIII's descendants, and James was seen as her most likely heir through his great-grandmother Margaret Tudor, who was Henry VIII's oldest sister. From 1601, in the last years of Elizabeth's life, certain English politicians—notably her chief minister Sir Robert Cecil—maintained a secret correspondence with James to prepare in advance for a smooth succession. With the Queen clearly dying, Cecil sent James a draft proclamation of his accession to the English throne in March 1603. Elizabeth died in the early hours of 24 March, and James was proclaimed king in London later the same day.

In Scotland, James was "James the sixth, King of Scotland", until 1604. He was proclaimed "James the first, King of England, France, and Ireland, defender of the faith" in London on 24 March 1603. On 20 October 1604, James issued a proclamation at Westminster changing his style to "King of Great Britain, France, and Ireland, Defender of the Faith, etc." The style was not used on English statutes, but was used on proclamations, coinage, letters, treaties, and in Scotland. James styled himself "King of France", in line with other monarchs of England between 1340 and 1800, although he did not actually rule France.

The co-operation between monarch and Parliament following the Gunpowder Plot was atypical. Instead, it was the previous session of 1604 that shaped the attitudes of both sides for the rest of the reign, though the initial difficulties owed more to mutual incomprehension than conscious enmity. On 7 July 1604, James had angrily prorogued Parliament after failing to win its support either for full union or financial subsidies. "I will not thank where I feel no thanks due", he had remarked in his closing speech. "... I am not of such a stock as to praise fools ... You see how many things you did not well ... I wish you would make use of your liberty with more modesty in time to come".

A dissident Catholic, Guy Fawkes, was discovered in the cellars of the parliament buildings on the night of 4–5 November 1605, the eve of the state opening of the second session of James's first English Parliament. He was guarding a pile of wood not far from 36 barrels of gunpowder with which Fawkes intended to blow up Parliament House the following day and cause the destruction, as James put it, "not only ... of my person, nor of my wife and posterity also, but of the whole body of the State in general". The sensational discovery of the Gunpowder Plot, as it quickly became known, aroused a mood of national relief at the delivery of the king and his sons. Salisbury exploited this to extract higher subsidies from the ensuing Parliament than any but one granted to Elizabeth. Fawkes and others implicated in the unsuccessful conspiracy were executed.

After the Gunpowder Plot, James sanctioned harsh measures to control non-conforming English Catholics. In May 1606, Parliament passed the Popish Recusants Act, which could require any citizen to take an Oath of Allegiance denying the Pope's authority over the king. James was conciliatory towards Catholics who took the Oath of Allegiance, and tolerated crypto-Catholicism even at court. Henry Howard, for Example, was a crypto-Catholic, received back into the Catholic Church in his final months. On ascending the English throne, James suspected that he might need the support of Catholics in England, so he assured the Earl of Northumberland, a prominent sympathiser of the old religion, that he would not persecute "any that will be quiet and give but an outward obedience to the law".

Under James the Plantation of Ulster by English and Scots Protestants began, and the English colonisation of North America started its course with the foundation of Jamestown, Virginia, in 1607, and Cuper's Cove, Newfoundland, in 1610. During the next 150 years, England would fight with Spain, the Netherlands, and France for control of the continent, while religious division in Ireland between Protestant and Catholic has lasted for 400 years. By actively pursuing more than just a personal union of his realms, he helped lay the foundations for a unitary British state.

As James's reign progressed, his government faced growing financial pressures, due partly to creeping inflation but also to the profligacy and financial incompetence of James's court. In February 1610, Salisbury proposed a scheme, known as the Great Contract, whereby Parliament, in return for ten royal concessions, would grant a lump sum of £600,000 to pay off the king's debts plus an annual grant of £200,000. The ensuing prickly negotiations became so protracted that James eventually lost patience and dismissed Parliament on 31 December 1610. "Your greatest error", he told Salisbury, "hath been that ye ever expected to draw honey out of gall". The same pattern was repeated with the so-called "Addled Parliament" of 1614, which James dissolved after a mere nine weeks when the Commons hesitated to grant him the money he required. James then ruled without parliament until 1621, employing officials such as the merchant Lionel Cranfield, who were astute at raising and saving money for the crown, and sold baronetcies and other dignities, many created for the purpose, as an alternative source of income.

When the Earl of Salisbury died in 1612, he was little mourned by those who jostled to fill the power vacuum. Until Salisbury's death, the Elizabethan administrative system over which he had presided continued to function with relative efficiency; from this time forward, however, James's government entered a period of decline and disrepute. Salisbury's passing gave James the notion of governing in person as his own chief Minister of State, with his young Scottish favourite Robert Carr carrying out many of Salisbury's former duties, but James's inability to attend closely to official Business exposed the government to factionalism.

In summer 1615, however, it emerged that Overbury had been poisoned. He had died on 15 September 1613 in the Tower of London, where he had been placed at the King's request. Among those convicted of the murder were Frances and Robert Carr, the latter having been replaced as the king's favourite in the meantime by Villiers. James pardoned Frances and commuted Carr's sentence of death, eventually pardoning him in 1624. The implication of the King in such a scandal provoked much public and literary conjecture and irreparably tarnished James's court with an image of corruption and depravity. The subsequent downfall of the Howards left Villiers unchallenged as the supreme figure in the government by 1619.

In Scotland, James attempted to bring the Scottish Kirk "so neir as can be" to the English church and to reestablish episcopacy, a policy that met with strong opposition from presbyterians. James returned to Scotland in 1617 for the only time after his accession in England, in the hope of implementing Anglican ritual. James's bishops forced his Five Articles of Perth through a General Assembly the following year, but the rulings were widely resisted. James left the church in Scotland divided at his death, a source of Future problems for his son.

In early 1623, Prince Charles, now 22, and Buckingham decided to seize the initiative and travel to Spain incognito, to win the infanta directly, but the mission proved an ineffectual mistake. The infanta detested Charles, and the Spanish confronted them with terms that included the repeal of anti-Catholic legislation by Parliament. Though a treaty was signed, the Prince and duke returned to England in October without the infanta and immediately renounced the treaty, much to the delight of the British people. Disillusioned by the visit to Spain, Charles and Buckingham now turned James's Spanish policy upon its head and called for a French match and a war against the Habsburg empire. To raise the necessary Finance, they prevailed upon James to call another Parliament, which met in February 1624. For once, the outpouring of anti-Catholic sentiment in the Commons was echoed in court, where control of policy was shifting from James to Charles and Buckingham, who pressured the king to declare war and engineered the impeachment of Lord Treasurer Lionel Cranfield, by now made Earl of Middlesex, when he opposed the plan on grounds of cost. The outcome of the Parliament of 1624 was ambiguous: James still refused to declare or fund a war, but Charles believed the Commons had committed themselves to Finance a war against Spain, a stance that was to contribute to his problems with Parliament in his own reign.

In early 1625, James was plagued by severe attacks of arthritis, gout, and fainting fits, and fell seriously ill in March with tertian ague and then suffered a stroke. He died at Theobalds House on 27 March during a violent attack of dysentery, with Buckingham at his bedside. James's funeral on 7 May was a magnificent but disorderly affair. Bishop John Williams of Lincoln preached the sermon, observing, "King Solomon died in Peace, when he had lived about sixty years ... and so you know did King James". The sermon was later printed as Great Britain's Salomon [sic].

Other influential anti-James histories written during the 1650s include: Sir Edward Peyton's Divine Catastrophe of the Kingly Family of the House of Stuarts (1652); Arthur Wilson's History of Great Britain, Being the Life and Reign of King James I (1658); and Francis Osborne's Historical Memoirs of the Reigns of Queen Elizabeth and King James (1658). David Harris Willson's 1956 biography continued much of this hostility. In the words of Historian Jenny Wormald, Willson's book was an "astonishing spectacle of a work whose every page proclaimed its author's increasing hatred for his subject". Since Willson, however, the stability of James's government in Scotland and in the early part of his English reign, as well as his relatively enlightened views on religion and war, have earned him a re-evaluation from many historians, who have rescued his reputation from this tradition of criticism.

Representative of the new historical perspective is the 2003 biography by Pauline Croft. Reviewer John Cramsie summarises her findings:

Some of James's biographers conclude that Esmé Stewart (later Duke of Lennox), Robert Carr (later Earl of Somerset), and George Villiers (later Duke of Buckingham) were his lovers. Sir John Oglander observed that he "never yet saw any fond husband make so much or so great dalliance over his beautiful spouse as I have seen King James over his favourites, especially the Duke of Buckingham" whom the King would, recalled Sir Edward Peyton, "tumble and kiss as a mistress." Restoration of Apethorpe Palace undertaken in 2004–08 revealed a previously unknown passage linking the bedchambers of James and Villiers.

James achieved more success in foreign policy. Never having been at war with Spain, he devoted his efforts to bringing the long Anglo–Spanish War to an end, and a peace treaty was signed between the two countries in August 1604, thanks to skilled diplomacy on the part of Robert Cecil and Henry Howard, now Earl of Northampton, which James celebrated by hosting a great banquet. Freedom of worship for Catholics in England, however, continued to be a major objective of Spanish policy, causing constant dilemmas for James, distrusted abroad for repression of Catholics while at home being encouraged by the Privy Council to show even less tolerance towards them.

The policy was supported by the Howards and other Catholic-leaning ministers and diplomats—together known as the Spanish Party—but deeply distrusted in Protestant England. When Sir Walter Raleigh was released from imprisonment in 1616, he embarked on a hunt for gold in South America with strict instructions from James not to engage the Spanish. Raleigh's expedition was a disastrous failure, and his son Walter was killed fighting the Spanish. On Raleigh's return to England, James had him executed to the indignation of the public, who opposed the appeasement of Spain. James's policy was further jeopardised by the outbreak of the Thirty Years' War, especially after his Protestant son-in-law, Frederick V, Elector Palatine, was ousted from Bohemia by the Catholic Emperor Ferdinand II in 1620, and Spanish troops simultaneously invaded Frederick's Rhineland home territory. Matters came to a head when James finally called a Parliament in 1621 to fund a military expedition in support of his son-in-law. The Commons on the one hand granted subsidies inadequate to Finance serious military operations in aid of Frederick, and on the other—remembering the profits gained under Elizabeth by naval attacks on Spanish gold shipments—called for a war directly against Spain. In November 1621, roused by Sir Edward Coke, they framed a petition asking not only for war with Spain but also for Prince Charles to marry a Protestant, and for enforcement of the anti-Catholic laws. James flatly told them not to interfere in matters of royal prerogative or they would risk punishment, which provoked them into issuing a statement protesting their rights, including freedom of speech. Urged on by the Duke of Buckingham and the Spanish ambassador Gondomar, James ripped the protest out of the record book and dissolved Parliament.

James was buried in Westminster Abbey. The position of the tomb was lost for many years until his lead coffin was found in the Henry VII vault in the 19th century, during an excavation.