Bothwell, under pretence of punishing some of the robber bands, mustered his forces, overcame the small guard that accompanied the queen on her journey from Stirling to Edinburgh, and carried off herself and Maitland as prisoners to Dunbar (19 April). That Bothwell acted in collusion with Mary is not proved, but despite the advice of her confessor, of the French representative, and of her best friends Mary agreed to go through a form of marriage with Bothwell. Her new husband was a Protestant, married already to the Earl of Huntly's sister from whom he had obtained a separation. The marriage ceremony was performed by the apostate Bishop of the Orkneys, who was soon to prove as disloyal to his queen as he had proved dishonest towards the Pope. Such a marriage celebrated under such circumstances created a most painful impression amongst the Catholics at home as well as in France and at Rome. It served to confirm their worst suspicions, and made them fear that Mary was about to desert the religion of her fathers. "With this act," wrote the papal ambassador who had been deputed to come to Scotland but who remained at Paris, "so dishonourable to herself, the propriety of sending any sort of envoy ceases unless indeed her Majesty, in order to amend her error and inspired by God, convert the Earl to the Catholic faith."[29]
Many of the lords, who had signed the bond to promote the marriage of Bothwell and Mary, professed to be shocked when they learned that the marriage had taken place. Relying upon the active intervention of Elizabeth they took up arms to avenge the murder of their king. The armies of the queen and of the lords met at Carbery Hill, where after some discussion Mary surrendered herself to the lords, and Bothwell was allowed to make his escape. The queen surrendered on the understanding that she was to be treated as queen, but she soon discovered that her captors intended to deprive her of her kingdom and possibly of her life. As a first step in the proceedings she was removed from Holyrood to Loch Leven (16th June). A document was drawn up embodying her abdication of the Scottish throne in favour of her infant son, and the appointment of her brother the Earl of Moray as regent during the minority. Until Moray's return the government was to be entrusted to a commission consisting of the Duke of Chatelherault, Lennox, Argyll, Atholl, Morton, Glencairn and Moray. Lord Lindsay and Sir Robert Melville were deputed to obtain the queen's signature, which they succeeded in obtaining only by threats and violence (24th July 1567). The young prince was crowned a few days later, John Knox acting as preacher on the occasion, and the apostate Bishop of the Orkneys as the chief minister. Steps were taken to ensure that Mary should not make her escape from imprisonment, and Bothwell who had fled to the Orkneys was forced to escape to Denmark, where he died in 1578. Moray hastened back from France, interviewed the queen at Loch Leven, accepted the office to which he had been appointed, and was proclaimed regent in Scotland. Severe measures were taken against the Catholic clergy many of whom fled from the kingdom. The queen's chapel at Holyrood was destroyed, and care was taken that the young king should be reared in the Protestant religion.
The lords of Scotland had taken up arms to avenge the murder of Darnley, but once they established themselves in power they took no steps to bring the murderers to justice, for the obvious reason that any judicial investigation must necessarily result in establishing their own guilt. Sir James Balfour, who had been involved deeply in the affair, was forgiven, on condition that he should surrender Edinburgh Castle into the hands of the regent. Parliament met in December 1567. It confirmed the abdication of the queen and the appointment of Moray. The laws passed against the Catholic Church in 1560 were renewed. It was enacted furthermore that for the future the kings and rulers of Scotland should swear to uphold the reformed religion and to extirpate heresy. The queen had demanded that she should be allowed to defend herself before Parliament against the attacks of her enemies, but the regent and council refused to comply with her request. Some of her friends, however, endeavoured to uphold her good name, and when they were defeated in Parliament they appealed to the people by publishing a defence of their sovereign.
Though every precaution was taken to ensure the safe-keeping of the queen, she succeeded in escaping from Loch Leven (2 May 1568). She was welcomed at Dunbar by the Primate of Scotland, the Hamiltons, Huntly, Argyll, Seaton, Cassillis, and others, and soon found herself at the head of an army of eight thousand men. She declared that her abdication having been secured by violence was worthless, and that the acts of the recent Parliament were null and void. She called upon all her loyal subjects to flock to her standard. The regent, aware that unless a sudden blow could be struck help would come to Mary from the Catholics of the north as well as from France and Spain, determined to take the field at once. The armies met at Langside, near Glasgow (13th May), where the forces of the queen were overthrown. Mary accompanied by a few faithful followers made her way south towards Galloway, and at last against the advice of her best friends she determined to cross the border to throw herself on the protection of the Queen of England.