Knox was acting as tutor to some young gentlemen in 1544-6 when he made the acquaintance of Wishart, the reformer, burned for heresy in the latter year by Cardinal Beaton, who three months later was murdered in revenge for this execution. The murderers held Beaton's Castle of St. Andrews against a siege and were there joined by Knox, who was 'called' to preach to them. In 1547, the castle capitulated to a French siege, and to the Regent of Scotland, and Knox was sent to the French galleys, where he remained for eighteen months. His labours with the oar could not have been severe, as he was able to write letters and theological pamphlets while on board. On being released, Knox came to England and held forth to a congregation of the reformed faith at Berwick. In 1551, he was made a Royal Chaplain, took part in the revision of the Prayer Book (1552) and was offered the Bishopric of Rochester, which he 'scrupled' and finally spurned. At the end of 1553 - after six months of Queen Mary - he fled to France with his wife, Marjory Bowes, whom he had married in the previous July. Early in 1554, he made his way to Geneva and Zurich, where he met Calvin and Bullinger. From one of these retreats, or from France, he issued, in July 1554, his 'Faithful Admonition to the Professors of God's Faith in England', directed against Queen Mary Tudor's Spanish marriage. At Frankfort, in the latter half of that year, Knox received a 'call' to preach to a congregation of English refugees, but got into dispute with a certain Dr. Cox and was obliged to return to Geneva. In 1555, he was again at Dieppe in France and thence made a nine months preaching tour of Scotland where, during his absence, the Reformation had made great strides. Knox now became acquainted with the leading Scottish nobles, whose zeal for Protestantism was quickened by the prospect of dividing the spoils of the old Church; and, during the same time, he fatally undermined the influence of the Regent, Mary of Guise. Whether increasing danger from her side, or a fresh 'call' from Geneva urged him, he quit Scotland in the Summer of 1556, and the trembling Scottish bishops were reduced to burning him in effigy. He remained principally in Geneva, maturing, at the feet of Calvin, his ideas of Church discipline until his final return to Scotland in 1559. Once at least, he was invited by the Scots lords to return, got as far as Dieppe and then thought better of it. From Geneva, he issued, in 1558, his 'First Blast of the Trumpet against the monstrous Regiment of Women' directed at the address of the Queens of England and Scotland and the Queen-Dowager-Regent of the latter country. He forgot that Elizabeth might soon be Queen of England. However, Elizabeth did not forget the 'First Blast'.

Finally, Knox came back to Scotland, in April 1559, organized the Protestant party for open war, preached publicly for the destruction of idols and idolatry and saw, before his eyes, the monuments of old Scottish art and piety hurled to the ground by the 'rascal multitude'. The Regent defended herself bravely to the last, but when Elizabeth, for her own safety's sake, was compelled, against her will, to send aid to the Scottish reformers, the Catholic cause in Scotland was as good as lost. With it, ended the life of the Regent and the old alliance of France and Scotland (1560). A Parliament in Edinburgh, in that year, established the new faith according to the Genevan model, although it must be said to Knox's credit that he always protested against the complete spoliation of the Church which accompanied the change. He would fain have saved decent stipends for the new 'presbyters' and ample funds to promote education; but he and the new Church were forced to pay the penalty of their alliance with the greedy nobles. Knox managed to get through a 'Book of Common Order', which was a kind of liturgy, but his next few years were spent in his famous struggle to put down the use of the mass in the chapel of Queen Mary who, after the death of her first husband, returned to her country as Queen-Dowager of France in August 1561. For four years, the great preacher and the able young Queen faced each other boldly, and the victory was not always with Knox. Had Mary's fatal passions not got the better of her judgement, she might have conciliated a moderate party even in a country divided by such savage family feuds. But after Mary's marriage, in 1565, the tragedy of Rizzio-Darnley-Bothwell rapidly unfolded itself and, in neither of the murders, can Knox's hand be traced. He was, in fact, absent in England when the King was killed and his next appearance is when he preached the coronation sermon for little James after his mother's abdication (1567). With the Regent Moray, Knox had not always lived upon good terms, but he now energetically supported his Government and did all that he could to prevent any temporizing with the exiled Queen. Indeed, he seems to have written to Cecil to beg him to put her to death. After the murder of Moray, Knox's political influence declined, but he still ruled the Church with a rod of iron. During his last years he was in constant ill health and used to get up from his bed only to preach. When once in the pulpit, however, his strength seemed to return and, at the end of the sermon, he seemed "lyk to ding that pulpit in blads and flee out of it". He died in his home in the High Street, Edinburgh, in 1572. His first wife had died in 1560 and, early in 1564, he had married Margaret Stuart, daughter of Lord Ochiltree.