Chalmers was ordained as minister of Kilmeny parish, Fife, in 1803. After reading William Wilberforce's Practical View of the Prevailing Religious System (1797), Chalmers adopted the evangelical position, which stressed the importance of faith for salvation. From 1815 he gained fame as one of the great pulpit orators in his ministry of Tron parish, Glasgow.

On becoming minister at St. John's, the largest and also the poorest parish in Glasgow, in 1819, Chalmers addressed himself to the problems of poverty. Receiving permission from the city to administer all the charitable funds donated in the churches, he had great success in ameliorating the condition of the poor while reducing costs. In 1823 he accepted the chair of moral philosophy at the University of St. Andrews, which he left five years later to become professor of divinity at the University of Edinburgh. At this time he was gaining recognition as a leader of the Evangelical party in the Church of Scotland, those who desired independence for the church from civil interference and who advocated the right of parishioners to choose their minister. The factional conflict culminated in the Disruption of 1843, when on May 18 a group of 203 commissioners walked out of the General Assembly of the Church of Scotland in protest against the government's refusal to grant spiritual independence to the church. Chalmers was made moderator of the new Free Church of Scotland. He was subsequently chosen as principal of the church's New College, founded in Edinburgh for ministerial training.

Chalmers was more concerned with the solution of human problems than with theological doctrines, and he sought to apply Christian ethics to economic issues. In An Enquiry into the Extent of and Stability of National Resources (1808) he argued that Napoleon's policy of continental blockade, far from ruining British trade, would merely cut off certain luxuries and turn to other, perhaps better, uses the funds that had supplied those luxuries. His work among the poor of Glasgow helped to fix his economic views, set forth in Christian and Civic Economy of Large Towns, 3 vol. (1821–26), and in On Political Economy (1832). His most significant theological study, On the Adaptation of External Nature to the Moral and Intellectual Constitution of Man, was written in 1833 and later incorporated in his Institutes of Theology (1849).