He and his family were Catholics and supporters of Mary, Queen of Scots. On 2 May 1568, he helped her escape from Loch Leven Castle and on 13 May fought for her at the defeat of Langside. His estates having been forfeited because of condemnation, Hamilton was concerned in the murder of the Regent James Stewart, 1st Earl of Moray in 1570, and also in that of the Regent Matthew Stewart, 4th Earl of Lennox in the following year; but in 1573 he recovered his estates.