He was the eldest son of Henry Whitgift, a merchant, of Great Grimsby, Lincolnshire, where he was born, probably between 1530 and 1533. The Whitgift family is thought to have originated in the relatively close Yorkshire village of Whitgift, adjoining the River Ouse.
John Whitgift's early education was entrusted to his uncle, Robert Whitgift, abbot of the neighbouring Wellow Abbey, on whose advice he was sent to St Anthony's School, London. In 1549 he matriculated at Queens' College, Cambridge, and in May 1550 he moved to Pembroke Hall, where the martyr John Bradford was his tutor. In May 1555 he became a fellow of Peterhouse.
Whitgift taught Francis Bacon and his older brother Anthony Bacon at Cambridge University in the 1570s. As their tutor, Whitgift bought the brothers their early classical text books, including works by Plato, Cicero and others.
Having taken holy orders in 1560, he became chaplain to Richard Cox, Bishop of Ely, who collated (that is, appointed) him to the rectory of Teversham, just to the east of Cambridge. In 1563 he was appointed Lady Margaret's Professor of Divinity at the University of Cambridge, and his lectures gave such satisfaction to the authorities that on 5 July 1566 they considerably augmented his stipend. The following year he was appointed Regius Professor of Divinity, and became master first of Pembroke Hall and then of Trinity. He had a principal share in compiling the statutes of the university, which passed the great seal on 25 September 1570, and in the November following he was chosen as vice-chancellor.
While at Cambridge he formed a close relationship with Andrew Perne, sometime vice-chancellor. Perne went on to live with Whitgift in his old age. Puritan satirists would later mock Whitgift as "Perne's boy" who was willing to carry his cloak-bag – thus suggesting that the two had enjoyed a homosexual relationship.
Whitgift's theological views were controversial. An aunt with whom he once lodged wrote that "though she thought at first she had received a saint into her house, she now perceived he was a devil". Macaulay's description of Whitgift as "a narrow, mean, tyrannical priest, who gained power by servility and adulation..." is, according to author of his 1911 Encyclopædia Britannica entry, "tinged with rhetorical exaggeration; but undoubtedly Whitgift's extreme High Church notions led him to treat the Puritans with exceptional intolerance". In a pulpit controversy with Thomas Cartwright regarding the constitutions and customs of the Church of England, his oratorical effectiveness proved inferior, but was able to exercise arbitrary authority: together with other heads of the university, he deprived Cartwright of his professorship, and in September 1571 Whitgift exercised his prerogative as master of Trinity to deprive him of his fellowship. In June of the same year Whitgift was nominated Dean of Lincoln. In the following year he published An Answere to a Certain Libel entitled an Admonition to the Parliament, which led to further controversy between the two churchmen. On 24 March 1577, Whitgift was appointed Bishop of Worcester, and during the absence of Sir Henry Sidney in Ireland in 1577 he acted as vice-president of Wales.
In August 1583 he was appointed Archbishop of Canterbury to replace Edmund Grindal, who had been placed under house arrest after his disagreement with Queen Elizabeth over 'prophesyings' and died in office. Whitgift placed his stamp on the church of the Reformation, and shared Elizabeth's hatred of Puritans. Although he wrote to Elizabeth remonstrating against the alienation of church property, Whitgift always retained her special confidence. In his policy against the Puritans and in his vigorous enforcement of the subscription test he thoroughly carried out her policy of religious uniformity.
He drew up articles aimed at nonconforming ministers, and obtained increased powers for the Court of High Commission. In 1586, he became a privy councillor. His actions gave rise to the Martin Marprelate tracts, in which the bishops and clergy were strongly opposed. His vigilance the printers of the tracts were discovered and punished, and to prevent the publication of such opinions he had the Act against Seditious Sectaries passed in 1593, making Puritanism an offence. In the controversy between Walter Travers and Richard Hooker, he prohibited the former from preaching, and he presented the latter with the rectory of Boscombe in Wiltshire, to help him complete his Ecclesiastical Polity, a work that in the end did not represent Whitgift's theological or ecclesiastical standpoints. In 1587, he had Welsh preacher John Penry brought before the High Commission, and imprisoned; Whitgift signed Penry's death warrant six years later.
In 1595, in conjunction with the Bishop of London and other prelates, he drew up the Calvinistic instrument known as the Lambeth Articles. Although the articles were signed and agreed by several bishops they were recalled by order of Elizabeth, claiming that the bishops had acted without her explicit consent. Whitgift maintained that she had given her approval.
Whitgift attended Elizabeth on her deathbed, and crowned James I. He was present at the Hampton Court Conference in January 1604, at which he represented eight bishops.
He died at Lambeth at the end of the following month. He was buried in Croydon at the Parish Church of St John Baptist (now Croydon Minster): his monument there with his recumbent effigy was practically destroyed when the church burnt down in 1867.
Whitgift is described by his biographer, Sir George Paule, as of "middle stature, strong and well shaped, of a grave countenance and brown complexion, black hair and eyes, his beard neither long nor thick." He left several unpublished works, included in the Manuscripts Angliae. Many of his letters, articles and injunctions are calendared in the published volumes of the State Papers series of the reign of Elizabeth. His Collected Works, edited for the Parker Society by John Ayre (3 vols., Cambridge, 1851–1853), include the controversial tracts mentioned above, two sermons published during his lifetime, a selection from his letters to Cecil and others, and some portions of his previously unpublished manuscripts.
Whitgift set up a charitable foundation, now The Whitgift Foundation, in Croydon, the site of a palace, a summer retreat of Archbishops of Canterbury. It supports homes for the elderly and infirm, and runs three independent schools – Whitgift School, founded in 1596, Trinity School of John Whitgift and, more recently, Old Palace School for girls, which is housed in the former Croydon Palace.
Whitgift Street near Lambeth Palace (the official London residence of the Archbishop of Canterbury) is named after him.
A comprehensive school in his home town of Grimsby, John Whitgift Academy, is named after him.
The Whitgift Centre, a major shopping centre in Croydon, is named after him. It is built on land still owned by the Whitgift Foundation.