Mary Stuart returned to Scotland in 1560. The Protestants were not happy having a Catholic queen and a religious riot took place soon after her arrival in Scotland. Mary attempted to reduce tension by accepting Protestants as her chief advisers. This included Hepburn who gradually became her closest friend in Scotland.

Elizabeth believed that Mary posed a threat to her throne. To counter this she suggested that her friend, Robert Dudley, Earl of Leicester, should marry Mary Stuart. Attempts were made to arrange this but in 1565 Mary married Henry Darnley, the son of Lady Margaret Douglas, the granddaughter of Henry VII. The marriage therefore strengthened her descendants' claim to the English throne.

In 1566 Mary gave birth to a son named James. The marriage was not a happy one and when Darnley was mysteriously killed while recovering from smallpox at Glasgow in January 1567, when the house in which he was in was blown up by gunpowder.

Suspicion fell on Hepburn. When Mary married Hepburn two months later, the Protestant lords rebelled against their queen. After Mary's army was defeated at Langside in 1567, Hepburn fled to Norway. He was seized by his enemies and taken to prison in Denmark. Never released, James Hepburn, Earl of Bothwell, died in chains in 1578.