James was son of King James IV of Scotland and his wife Margaret Tudor, a daughter of Henry VII of England and sister of Henry VIII, and was the only legitimate child of James IV to survive infancy. He was born on 10 April 1512 at Linlithgow Palace, Linlithgowshire and baptized the following day, receiving the titles Duke of Rothesay and Prince and Great Steward of Scotland. He became king at just seventeen months old when his father was killed at the Battle of Flodden Field on 9 September 1513.

James was crowned in the Chapel Royal at Stirling Castle on 21 September 1513. During his childhood the country was ruled by regents, first by his mother, until she remarried the following year, and then by John Stewart, 2nd Duke of Albany, next in line to the Crown after James and his younger brother, the posthumously-born Alexander Stewart, Duke of Ross, who died in infancy. Other regents included Robert Maxwell, 5th Lord Maxwell, a member of the Council of Regency who was also bestowed as Regent of Arran, the largest island in the Firth of Clyde. In February 1517 James came from Stirling to Holyroodhouse, Edinburgh, but during an outbreak of plague in the city he was moved to the care of Antoine d'Arces at nearby rural Craigmillar Castle. At Stirling, the 10-year-old James had a guard of 20 footmen dressed in his colours, red and yellow. When he went to the park below the Castle, "by secret and in right fair and soft wedder (weather)," six horsemen would scour the countryside two miles roundabout for intruders. Poets wrote their own nursery rhymes for James and advised him on royal behavior. As a youth, his education was in the care of University of St Andrews poets such as Sir David Lyndsay. william Stewart, in his poem Princelie Majestie, counselled James against ice-skating:

As early as August 1517 a clause of the Treaty of Rouen provided that if the Auld Alliance between France and Scotland was maintained, James should have a French royal bride. Yet the daughters of Francis I of France were promised elsewhere or sickly. Perhaps to remind Francis of his obligations James's envoys began negotiations for his marriage elsewhere from the summer of 1529, both to Catherine de' Medici, the Duchess of Urbino, and Mary of Austria, Queen of Hungary, the sister of Holy Roman Emperor Charles V. But plans changed. In February 1533, two French ambassadors, Guillaume du Bellay, sieur de Langes, and Etienne de Laigue, sieur de Beauvais, who had just been in Scotland, told the Venetian ambassador in London that James was thinking of marrying Christina of Denmark. Marguerite d'Angoulême, sister of Francis I, suggested her sister-in-law Isabella, who was the same age.

In the autumn of 1524 James dismissed his regents and was proclaimed an adult ruler by his mother. Several new court servants were appointed including a trumpeter, Henry Rudeman. Thomas Magnus, the English diplomat, gave an impression of the new Scottish court at Holyroodhouse on All Saints' Day 1524: "trumpets and shamulles did sounde and blewe up mooste pleasauntely." Magnus saw the young king singing, playing with a spear at Leith, and with his horses, and he was given the impression that the king preferred English manners over French fashions.

In 1525 Archibald Douglas, 6th Earl of Angus, the young king's stepfather, took custody of James and held him as a virtual prisoner for three years, exercising power on his behalf. There were several attempts made to free the young King – one by Walter Scott of Branxholme and Buccleuch, who ambushed the King's forces on 25 July 1526 at the battle of Mel Rose, and was routed off the field. Another attempt later that year, on 4 September at the battle of Linlithgow Bridge, failed again to relieve the King from the clutches of Angus. When James and his mother came to Edinburgh on 20 November 1526, she stayed in the chambers at Holyroodhouse, which Albany had used, James using the rooms above. In February 1527 Henry Fitzroy, Duke of Richmond, gave James twenty hunting hounds and a huntsman. Magnus thought the Scottish servant sent to Sheriff Hutton Castle for the dogs was intended to note the form and fashion of the Duke's household, for emulation in Scotland. James finally escaped from Angus's care in 1528 and assumed the reins of government himself.

As a patron of poets and authors James supported william Stewart and John Bellenden, the son of his nurse, who translated the Latin History of Scotland compiled in 1527 by Hector Boece into verse and prose. Sir David Lindsay of the Mount, the Lord Lyon, head of the Lyon Court and diplomat, was a prolific poet. He produced an interlude at Linlithgow Palace thought to be a version of his play The Thrie Estaitis in 1540. James also attracted the attention of international authors. The French poet Pierre de Ronsard, who had been a page of Madeleine of Valois, offered unqualified praise;

Domestic and international policy was affected by the Reformation, especially after Henry VIII broke from the Catholic Church. James V did not tolerate heresy, and during his reign a number of outspoken Protestants were persecuted. The most famous of these was Patrick Hamilton, who was burned at the stake as a heretic at St Andrews in 1528. Later in the reign, the English ambassador Ralph Sadler tried to encourage James to close the monasteries and take their revenue so that he would not have to keep sheep like a mean subject. James replied that he had no sheep, he could depend on his god-father the King of France, and it was against reason to close the abbeys that "stand these many years, and God's Service maintained and kept in the same, and I might have anything I require of them." (Sadler knew that James did farm sheep on his estates.)

Many of the sons of his aristocratic mistresses entered ecclesiastical careers. Pope Clement VII sent a dispensation to James V dated 30 August 1534, allowing four of the children to take holy orders when they came of age. The document stated that James elder was in his fifth year, James younger and John in their third year, and Robert in his first year.

Francis I insisted that his daughter Madeleine's health was too poor for marriage. Eventually, on 6 March 1536, a contract was made for James V to marry Mary of Bourbon, daughter of the Duke of Vendôme. She would have a dowry as if she were a French Princess. James decided to visit France in person. He sailed from Kirkcaldy on 1 September 1536, with the Earl of Argyll, the Earl of Rothes, Lord Fleming, David Beaton, the Prior of Pittenweem, the Laird of Drumlanrig and 500 others, using the Mary Willoughby as his flagship. First he visited Mary of Bourbon at St. Quentin in Picardy, but then went south to meet King Francis I. During his stay in France, in October 1536, James went boar-hunting at Loches with Francis, his son the Dauphin, the King of Navarre and Ippolito II d'Este.

James and Madeleine returned from France on 19 May 1537, arriving at Leith, the king's Scottish fleet accompanied with ten great French ships. As the couple sailed northwards, some Englishmen had come aboard off Bridlington and Scarborough. While the fleet was off Bamburgh on 15 May, three English fishing boats supplied fish, and the King's butcher landed in Northumbria to buy meat. The English border authorities were dismayed by this activity.

One technological initiative was a special mill for polishing armour at Holyroodhouse next to his mint. The mill had a pole drive 32 feet long powered by horses. Mary of Guise's mother Antoinette of Bourbon sent him an armourer. The armourer made steel plates for his jousting saddles in October 1538, and delivered a skirt of plate armour in February 1540. In the same year, for his wife's coronation, the treasurer's accounts record that James personally devised fireworks made by his master gunners. When James took steps to suppress the circulation of slanderous ballads and rhymes against Henry VIII, Henry sent Fulke ap Powell, Lancaster Herald, to give thanks and to make arrangements for the present of a lion for James's menagerie of exotic pets.

James increased his income by tightening control over royal estates and from the profits of justice, customs and feudal rights. He also gave his illegitimate sons lucrative benefices, diverting substantial church wealth into his coffers. James spent a large amount of his wealth on building work at Stirling Castle, Falkland Palace, Linlithgow Palace and Holyrood, and he built up a collection of tapestries from those inherited from his father. James sailed to France for his first marriage and strengthened the royal fleet. In 1540 he sailed to Kirkwall in Orkney, then Lewis, in his ship the Salamander, first making a will in Leith, knowing this to be "uncertane aventuris." The purpose of this voyage was to show the royal presence and hold regional courts, called "justice ayres."

The death of James's mother in 1541 removed any incentive for peace with England, and war broke out. Initially the Scots won a victory at the Battle of Haddon Rig in August 1542. The Imperial ambassador in London, Eustace Chapuys, wrote on 2 October that the Scottish ambassadors ruled out a conciliatory meeting between James and Henry VIII in England until the pregnant Mary of Guise delivered her child. Henry would not accept this condition and mobilised his army against Scotland.

James was with his army at Lauder on 31 October 1542. Although he hoped to invade England, his nobles were reluctant. He returned to Edinburgh, on the way writing a letter in French to his wife from Falahill mentioning he had three days of illness. The next month his army suffered a serious defeat at the Battle of Solway Moss. He took ill shortly after this, on 6 December; by some accounts this was a nervous collapse caused by the defeat, and he may have died from the grief, although some historians consider that it may just have been an ordinary fever. John Knox later described his final movements in Fife.

James was succeeded by his infant daughter Mary. He was buried at Holyrood Abbey alongside his first wife Madeleine and his two sons in January 1543. David Lindsay supervised the construction of his tomb. One of his French artists, Andrew Mansioun, carved a lion and an inscription in Roman letters measuring eighteen feet. The tomb was destroyed in the sixteenth century, according to william Drummond of Hawthornden as early as 1544, by the English during the burning of Edinburgh. Scotland was ruled by Regent Arran and was soon drawn into the war of the Rough Wooing.

According to legend James was nicknamed "King of the Commons" as he would sometimes travel around Scotland disguised as a Common man, describing himself as the "Gudeman of Ballengeich" ('Gudeman' means 'landlord' or 'farmer', and 'Ballengeich' was the nickname of a road next to Stirling Castle – meaning 'windy pass' in Gaelic). James was also a keen lute player. In 1562 Sir Thomas Wood reported that James had "a singular good ear and could sing that he had never seen before" (sight-read), but his voice was "rawky" and "harske." At court, James maintained a band of Italian Musicians who adopted the name Drummond. These were joined for the winter of 1529/30 by a musician and diplomat sent by the Duke of Milan, Thomas de Averencia de Brescia, probably a lutenist. The Historian Andrea Thomas makes a useful distinction between the loud music provided at ceremonies and processionals and instruments employed for more private occasions or worship; the music fyne described by Helena Mennie Shire. This quieter music included a consort of viols played by four Frenchmen led by Jacques Columbell. It seems certain that David Peebles wrote music for James V and probable that the Scottish Composer Robert Carver was in royal employ, though evidence is lacking.