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He was apparently one of the Cambridge men who gathered at the White Horse Tavern for Bible-reading and theological discussion early in the third decade of the 16th century. At the Christmas Midnight Mass at St Edward's Church in 1525, Barnes gave an openly evangelical sermon proclaiming the gospel and accusing the Church of its heresies, now sometimes considered to be the first sermon of the English Reformation.  As a result, in 1526 he was brought before the vice-chancellor for preaching a heterodox sermon, and was subsequently examined by Wolsey and four other bishops. He was condemned to abjure or be burnt; and preferring the former alternative, was committed to the Fleet prison and afterwards to the Austin Friars in London.
He escaped to Antwerp in 1528, and also visited Wittenberg, where he made Martin Luther's acquaintance. He also came across Stephen Vaughan, an agent of Thomas Cromwell and an advanced reformer, who recommended him to Cromwell: "Look well," he wrote, "upon Dr Barnes' book. It is such a piece of work as I have not yet seen any like it. I think he shall seal it with his blood" (Letters and Papers of Henry VIII).
In 1531 Barnes returned to England, and became one of the chief intermediaries between the English government and Lutheran Germany. In 1535 he was sent to Germany, in the hope of inducing Lutheran divines to approve of Henry's divorce from Catherine of Aragon, and four years later he was employed in negotiations connected with Anne of Cleves's marriage. The policy was Cromwell's, but Henry VIII had already in 1538 refused to adopt Lutheran theology, and the statute of Six Articles (1539), followed by the king's disgust with Anne of Cleves (1540), brought the agents of that policy to ruin.
An attack upon Bishop Gardiner by Barnes in a sermon at St Paul's Cross was the signal for a bitter struggle between the Protestant and reactionary parties in Henry's council, which raged during the spring of 1540. Barnes was forced to apologize and recant; and Gardiner delivered a series of sermons at St Paul's Cross to counteract Barnes' invective. But a month or so later Cromwell was made earl of Essex, Gardiner's friend, Bishop Sampson, was sent to the Tower, and Barnes reverted to Lutheranism. It was a delusive victory. In July, Cromwell was attainted, Anne of Cleves was divorced and Barnes was burnt (30 July 1540).
Barnes was one of six executed on the same day: two, William Jerome and Thomas Gerrard, were, like himself, burnt for heresy under the Six Articles; three, Thomas Abel, Richard Fetherstone and Edward Powell, were hanged for treason in denying the royal supremacy. Both Lutherans and Catholics on the continent were shocked. Luther published Barnes' confession with a preface of his own as Bekenntnis des Glaubens (1540).