His father died in 1576, when Donne was four years old, leaving his son fatherless and his widow, Elizabeth Heywood, with the responsibility of raising their children alone. Heywood was also from a recusant Roman Catholic family, the daughter of John Heywood, the Playwright, and sister of the Reverend Jasper Heywood, a Jesuit priest and translator. She was a great-niece of the Roman Catholic martyr Thomas More. This tradition of martyrdom would continue among Donne's closer relatives, many of whom were executed or exiled for religious reasons. Donne was educated privately; however, there is no evidence to support the popular claim that he was taught by Jesuits. Donne's mother married Dr. John Syminges, a wealthy widower with three children, a few months after Donne's father died. Donne thus acquired a stepfather. Two more of his sisters, Mary and Katherine, died in 1581. Donne's mother lived her last years in the Deanery after Donne became Dean of St Paul's, and died just two months before Donne, in January 1631. In 1583, the 11-year-old Donne began studies at Hart Hall, now Hertford College, Oxford. After three years of studies there, Donne was admitted to the University of Cambridge, where he studied for another three years. However, Donne could not obtain a degree from either institution because of his Catholicism, since he refused to take the Oath of Supremacy required to graduate.

In 1591 Donne was accepted as a student at the Thavies Inn legal school, one of the Inns of Chancery in London. On 6 May 1592 he was admitted to Lincoln's Inn, one of the Inns of Court. In 1593, five years after the defeat of the Spanish Armada and during the intermittent Anglo-Spanish War (1585–1604), Queen Elizabeth issued the first English statute against sectarian dissent from the Church of England, titled "An Act for restraining Popish recusants". It defined "Popish recusants" as those "convicted for not repairing to some Church, Chapel, or usual place of Common Prayer to hear Divine Service there, but forbearing the same contrary to the tenor of the laws and statutes heretofore made and provided in that behalf". Donne's brother Henry was also a university student prior to his arrest in 1593 for harbouring a Catholic priest, william Harrington, whom he betrayed under torture. Harrington was tortured on the rack, hanged until not quite dead, and then subjected to disembowelment. Henry Donne died in Newgate Prison of bubonic plague, leading Donne to begin questioning his Catholic faith.

During his lifetime several likenesses were made of the poet. The earliest was the anonymous portrait of 1594 now in the National Portrait Gallery, London which has been recently restored. One of the earliest Elizabethan portraits of an author, the fashionably dressed poet is shown darkly brooding on his love. The portrait was described in Donne's will as "that picture of myne wych is taken in the shaddowes", and bequeathed by him to Robert Kerr, 1st Earl of Ancram. Other paintings include a 1616 head and shoulders after Isaac Oliver, also in the National Portrait Gallery, and a 1622 head and shoulders in the Victoria and Albert Museum. In 1911 the young Stanley Spencer devoted a visionary painting to John Donne arriving in heaven (1911) which is now in the Fitzwilliam Museum.

During and after his education, Donne spent much of his considerable inheritance on women, literature, pastimes and travel. Although no record details precisely where Donne travelled, he did cross Europe and later fought with the Earl of Essex and Sir Walter Raleigh against the Spanish at Cadiz (1596) and the Azores (1597), and witnessed the loss of the Spanish flagship, the San Felipe. According to Izaak Walton, who wrote a biography of Donne in 1658:

During the next four years Donne fell in love with Egerton's niece Anne More, and they were secretly married just before Christmas in 1601, against the wishes of both Egerton and George More, who was Lieutenant of the Tower and Anne's father. Upon discovery, this wedding ruined Donne's career, getting him fired and put in Fleet Prison, along with the Church of England priest Samuel Brooke, who married them, and the man who acted as a witness to the wedding. Donne was released shortly thereafter when the marriage was proven valid, and he soon secured the release of the other two. Walton tells us that when Donne wrote to his wife to tell her about losing his post, he wrote after his name: John Donne, Anne Donne, Un-done. It was not until 1609 that Donne was reconciled with his father-in-law and received his wife's dowry.

In 1602 John Donne was elected as Member of Parliament (MP) for the constituency of Brackley, but membership was not a paid position. Queen Elizabeth I died in 1603, being succeeded by King James VI of Scotland as King James I of England. The fashion for coterie poetry of the period gave Donne a means to seek patronage, and many of his poems were written for wealthy friends or patrons, especially MP Sir Robert Drury of Hawsted (1575–1615), whom he met in 1610 and became Donne's chief patron, furnishing him and his family an apartment in his large house in Drury Lane.

After his release, Donne had to accept a retired country life in a small house in Pyrford, Surrey, owned by Anne's cousin, Sir Francis Wooley, where they resided until the end of 1604. In spring 1605 they moved to another small house in Mitcham, London, where he scraped a meager living as a Lawyer, while Anne Donne bore a new baby almost every year. Though he also worked as an assistant pamphleteer to Thomas Morton writing anti-Catholic pamphlets, Donne was in a constant state of financial insecurity.

There were musical settings of Donne's lyrics even during his lifetime. These included Alfonso Ferrabosco the younger's ("So, so, leave off this last lamenting kisse" in his 1609 Ayres); John Cooper's ("The Message"); Henry Lawes' ("Break of Day"); John Dowland's ("Break of Day" and "To ask for all thy love"); and settings of "A Hymn to God the Father" by John Hilton the younger and Pelham Humfrey (1688). After the 17th century there were no more until the start of the 20th century with Havergal Brian ("A nocturnal on St Lucy's Day", first performed in 1905), Eleanor Everest Freer ("Break of Day, published in 1905) and Walford Davies ("The Cross", 1909) among the earliest. In 1945, Benjamin Britten set nine of Donne's Holy Sonnets in his song cycle for voice and piano The Holy Sonnets of John Donne. Lieder.net currently lists 162 settings of 62 texts by Donne. Among them is also the choral setting of "Negative Love" that opens Harmonium (1981) by John Adams..

In 1610 and 1611 Donne wrote two anti-Catholic polemics: Pseudo-Martyr and Ignatius His Conclave for Morton. He then wrote two Anniversaries, An Anatomy of the World (1611) and Of the Progress of the Soul (1612) for Drury. Although James was pleased with Donne's work, he refused to reinstate him at court and instead urged him to take holy orders. At length, Donne acceded to the king's wishes, and in 1615 was ordained priest the Church of England.

Some have speculated that Donne's numerous illnesses, financial strain, and the deaths of his friends all contributed to the development of a more somber and pious tone in his later poems. The change can be clearly seen in "An Anatomy of the World" (1611), a poem that Donne wrote in memory of Elizabeth Drury, daughter of his patron, Sir Robert Drury of Hawstead, Suffolk. This poem treats Elizabeth's demise with extreme gloominess, using it as a symbol for the Fall of Man and the destruction of the universe.

In 1615 Donne was awarded an honorary doctorate in divinity from Cambridge University, and became a Royal Chaplain in the same year, and a Reader of Divinity at Lincoln's Inn in 1616, where he served in the chapel as minister until 1622. In 1618 he became chaplain to Viscount Doncaster, who was on an embassy to the princes of Germany. Donne did not return to England until 1620. In 1621 Donne was made Dean of St Paul's, a leading and well-paid position in the Church of England, which he held until his death in 1631. During his period as dean his daughter Lucy died, aged eighteen. In late November and early December 1623 he suffered a nearly fatal illness, thought to be either typhus or a combination of a cold followed by a period of fever. During his convalescence he wrote a series of meditations and prayers on health, pain, and sickness that were published as a book in 1624 under the title of Devotions upon Emergent Occasions. One of these meditations, Meditation XVII, later became well known for its phrases "No man is an Iland" (often modernised as "No man is an island") and "...for whom the bell tolls". In 1624 he became vicar of St Dunstan-in-the-West, and 1625 a prolocutor to Charles I. He earned a reputation as an eloquent preacher and 160 of his sermons have survived, including the famous Death’s Duel sermon delivered at the Palace of Whitehall before King Charles I in February 1631.

The poem "A Nocturnal upon S. Lucy's Day, Being the Shortest Day", concerns the poet's despair at the death of a loved one. In it Donne expresses a feeling of utter negation and hopelessness, saying that "I am every dead thing ... re-begot / Of absence, darkness, death." This famous work was probably written in 1627 when both Donne's friend Lucy, Countess of Bedford, and his daughter Lucy Donne died. Three years later, in 1630, Donne wrote his will on Saint Lucy's Day (13 December), the date the poem describes as "Both the year's, and the day's deep midnight".

Towards the end of his life Donne wrote works that challenged death, and the fear that it inspired in many men, on the grounds of his belief that those who die are sent to Heaven to live eternally. One Example of this challenge is his Holy Sonnet X, "Death Be Not Proud", from which come the famous lines "Death, be not proud, though some have called thee / Mighty and dreadful, for thou art not so." Even as he lay dying during Lent in 1631, he rose from his sickbed and delivered the Death's Duel sermon, which was later described as his own funeral sermon. Death's Duel portrays life as a steady descent to suffering and death; death becomes merely another process of life, in which the 'winding sheet' of the womb is the same as that of the grave. Hope is seen in salvation and immortality through an embrace of God, Christ and the Resurrection.

His work has received much criticism over the years, especially concerning his metaphysical form. Donne is generally considered the most prominent member of the metaphysical poets, a phrase coined in 1781 by Samuel Johnson, following a comment on Donne by John Dryden. Dryden had written of Donne in 1693: "He affects the metaphysics, not only in his satires, but in his amorous verses, where nature only should reign; and perplexes the minds of the fair sex with nice speculations of philosophy, when he should engage their hearts, and entertain them with the softnesses of love." In Life of Cowley (from Samuel Johnson's 1781 work of biography and criticism Lives of the Most Eminent English Poets), Johnson refers to the beginning of the seventeenth century in which there "appeared a race of Writers that may be termed the metaphysical poets". Donne's immediate successors in poetry therefore tended to regard his works with ambivalence, with the Neoclassical poets regarding his conceits as abuse of the metaphor. However he was revived by Romantic poets such as Coleridge and Browning, though his more recent revival in the early twentieth century by poets such as T. S. Eliot and critics like F R Leavis tended to portray him, with approval, as an anti-Romantic.

Prose texts by Donne have also been set to music. In 1954, Priaulx Rainier set some in her Cycle for Declamation for solo voice. In 2009, the American Jennifer Higdon composed the choral piece On the Death of the Righteous, based on Donne's sermons. Still more recent is the Russian minimalist Anton Batagov's " I Fear No More, selected songs and meditations of John Donne" (2015).

Beginning in the 20th century, several historical novels appeared taking as their subject various episodes in Donne's life. His courtship of Anne More is the subject of Elizabeth Gray Vining's Take Heed of Loving Me: A novel about John Donne (1963) and Maeve Haran's The Lady and the Poet (2010). Both characters also make interspersed appearances in Mary Novik's Conceit (2007), where the main focus is on their rebellious daughter Pegge. English treatments include Garry O'Connor's Death's Duel: a novel of John Donne (2015), which deals with the poet as a young man. He also plays a significant role in Christie Dickason's The Noble Assassin (2012), a novel based on the life of Donne's patron and (the author claims) his lover, Lucy Russell, Countess of Bedford. Finally there is Bryan Crockett's Love's Alchemy: a John Donne Mystery (2015), in which the poet, blackmailed into Service in Robert Cecil's network of spies, attempts to avert political disaster and at the same time outwit Cecil.

There have been settings in popular music as well. One is the version of the song "Go and Catch a Falling Star" on John Renbourn's debut album John Renbourn (1966), in which the last line is altered to "False, ere I count one, two, three". On their 1992 album Duality, the English Neoclassical Dark Wave band In The Nursery used the entirety of Donne's "A Valediction: Forbidding Mourning" for the track "Mecciano" and an augmented version of "A Fever" for the track "Corruption."

Some scholars believe that Donne's literary works reflect the changing trends of his life, with love poetry and satires from his youth and religious sermons during his later years. Other scholars, such as Helen Gardner, question the validity of this dating—most of his poems were published posthumously (1633). The exception to these is his Anniversaries, which were published in 1612 and Devotions upon Emergent Occasions published in 1624. His sermons are also dated, sometimes specifically by date and year.