By late 1545 Knox had attached himself closely to the reformer George Wishart. When, after Wishart's execution (1546), a group of Protestant conspirators took revenge by murdering Cardinal David Beaton, Knox, now definitely a Protestant, took refuge with them in St. Andrews Castle and preached in the parish church. Attacked by both Scottish and French forces, the castle was eventually surrendered (1547), and Knox served 19 months in the French galleys before his release (1549) through the efforts of the English government of Edward VI.

Knox spent the next few years in England, preaching in Berwick and Newcastle as a licensed minister of the crown and serving briefly as a royal chaplain. He helped to prepare the second Book of Common Prayer, but he declined a bishopric in the newly established Church of England.

Shortly after the accession (1553) of the Catholic Mary I to the English throne, Knox went into exile on the Continent, living chiefly in Geneva and Frankfurt. In Geneva he consulted with John Calvin on questions of church doctrine and civil authority.

Meanwhile, through his frequent letters, he exerted considerable influence among Protestants in England and Scotland; in his "Faithful Admonition" pamphlet of 1554 he began to urge the duty of the righteous to overthrow "ungodly" monarchs. In 1555-56 he visited Scotland, preaching in private and counseling the Protestant congregations. After his return to Geneva, where he served (1556-58) as pastor to the English congregation, he wrote the First Blast of the Trumpet against the Monstrous Regiment [i.e., regimen] of Women. That fiery tract was directed against the Catholic Mary of Guise, regent of Scotland, and Queen Mary of England, but it also alienated the Protestant Elizabeth I, who succeeded to the English throne in 1558.

In 1557 the Scottish Protestant nobles signed their First Covenant, banding together to form the group known as the lords of the congregation (see Scotland, Church of). When, in 1559, Mary of Guise moved against the Protestants, the lords of the congregation took up arms and invited Knox back from Geneva to lead them. Aided by England and by the regent's death in 1560, the reformers forced the withdrawal of the French troops that had come to Mary's aid and won their freedom as well as dominance for the new religion.

Under Knox's direction, a confession of faith (basically Calvinist) was drawn up (1560) and passed by the Scottish Parliament, which also passed laws abolishing the authority of the pope and condemning all creeds and practices of the old religion. The Book of Discipline, however, which provided an organizational structure for the new church, failed to get adequate approval from the nobles in 1561.

When Mary Queen of Scots arrived from France to assume her crown in the same year, many Protestant lords deserted Knox and his cause, and some even joined the queen. From his pulpit and in personal debates with Mary on questions of theology and the loyalty owed by the subject to his monarch, Knox stubbornly defied Mary's authority and thundered against her religion. The queen's marriage to Lord Darnley, her suspected complicity in his murder, and her hasty marriage to James Hepburn, earl of Bothwell, stirred the Protestant lords to revolt. Mary was forced to abdicate (1567) in favor of her young son, James VI. All the acts of 1560 were then confirmed, thereby establishing Presbyterianism as the official religion.

Despite the ill health of his last years, Knox continued to be an outspoken preacher until his death. It has been said of Knox that "rarely has any country produced a stronger will." His single-minded zeal made him the outstanding leader of the Scottish Reformation and an important influence on the Protestant movements in England and on the Continent, but the same quality tended to close his mind to divergent views. His History of the Reformation in Scotland, finished in 1564 but published in 1584 after his death, is a striking record of that conflict, but includes a number of misstatements and omissions resulting from his strong bias.