In December 1557 he was one of the leaders of the Reform Party who subscribed to the memorable Covenant which had been drawn up for the support and defence of the Protestant religion, and who thenceforth assumed the name the Lords of the Congregation.
In 1559, in consequence of the rigorous proceedings against Protestants by the Queen Regent, he and his relative, Sir Hugh Campbell of Loudon, the Sheriff of Ayr, requested an audience of Her Majesty, at which they reminded her of her promises of religious toleration.
On the Queen's replying that "promises ought not to be urged upon princes, unless they can conveniently fulfil them"; "then", they said, "since you are resolved to keep no faith with your subjects we will renounce our allegiance", an answer which induced her to stop her proceedings.
When Mary, Queen of Scots, was taken to Lochleven Castle that month, Glencairn hastened with his domestics to the Chapel Royal of Holyroodhouse and destroyed all the sacred images, demolished the altar, tore down the pictures, and defaced all the ornaments.
A satirical poem against the Popish Party, entitled The Hermit of Allareit or Loretto, near Musselburgh, written by Lord Glencairn, and preserved in Knox's History of the Reformation, is also found in Sibbald's Chronicle of Scottish Poetry.