The illegitimate son of a parson at Kingussie, Inverness-shire, Leslie studied at the universities of Aberdeen, Paris, and Poitiers. From about 1554 he taught canon law at King's College, Aberdeen, and held a diocesan administrative post. When Mary Stuart, the recently widowed queen consort of France, returned to reign in Scotland in 1561, Leslie became her adviser, holding judicial office, a privy councillorship, and (from 1566) the bishopric of Ross. Unhappy with Mary's attachment to the Earl of Bothwell, he accused the Earl of seducing her with black magic. Leslie was loyal to Mary even after her forced abdication of the Scottish throne. He tried to defend her before the board of inquiry convened at York (Oct. 4, 1568) by Queen Elizabeth, and in 1569 he became Mary's accredited representative at Elizabeth's court.

Leslie was implicated in an unsuccessful revolt in the north of England in January 1569 but was acquitted. He then joined Roberto Ridolfi (Ridolfi, Roberto), a Florentine businessman living in London, in planning a more ambitious rebellion. Elizabeth was to be deposed (and murdered) in favour of Mary, with the aid of Spanish armed forces; Mary was to be married to Thomas Howard, 4th duke of Norfolk. Leslie's confessions (October–November 1571) and other evidence led to Norfolk's execution for treason (June 2, 1572) and to his own imprisonment. Released late in 1573, Leslie tried without success to obtain assistance for Mary from continental rulers. From 1579 he lived in France, where he was suffragan and vicar general of the diocese of Rouen. At Rome in 1578 Leslie published his history of Scotland, De origine, moribus et rebus gestis Scotorum. Partly derived from the works of Hector Boece and John Major, it presents a strongly Catholic viewpoint.