Dauphiné was one of the first of the provinces of France to be touched by the revolutionary ideals, and Barnave was one of the first to give voice to the general feeling, in a pamphlet entitled Esprit des édüs enregistrés militairement le 20 mai 1788. He was immediately elected deputy, with his father, to the states of Dauphiné, and took a prominent part in their debates.
He soon rose to prominence in the National Assembly, becoming the friend of most of the leaders of the party originating in the Third Estate, and formed with Adrien Duport and Alexandre Lameth the group known during the Constituent Assembly as "the triumvirate". He took part in the conference on the claims of the three orders, drew up the first address to King Louis XVI, and supported the proposal of Emmanuel Joseph Sieyès that the Assembly should declare itself "National". Until 1791, he was one of the main members of the club known later as the Jacobins, of which he drew up the manifesto and first rulebook.
His conflict with Mirabeau on the question of assigning to the King the right to make peace or war (from May 16 to 23, 1791) was one of the main episodes of the Assembly's mandate. In August 1790, after a vehement debate, he fought a duel with Jacques Antoine Marie de Cazalès, in which the latter was slightly wounded. About the close of October 1790, Barnave was called to the presidency of the Assembly. On the death of Mirabeau a few months later, Barnave paid a high tribute to his worth and public services, designating him the "William Shakespeare of oratory".
Barnave led the Feuillants out of the Jacobin Club in early 1791, and their faction entered a conflict with the Girondists after they opposed war with Habsburgs, and were driven out of the Assembly. His public career came to an end, and he returned to Grenoble at the beginning of 1792. His sympathy and relations with the royal family, to whom he had submitted a plan for a counter-revolution, and his desire to check the violence of the Revolution, brought on him suspicion of treason.
He was denounced (August 15, 1792) in the Legislative Assembly, arrested and imprisoned for ten months at Grenoble, then transferred to Fort Barraux, and in November 1793 to Paris (during the Reign of Terror). On November 28 he appeared before the Revolutionary Tribunal. He was condemned on the evidence of papers found in the Tuileries Palace, and guillotined the next day, alongside Marguerite-Louis-François Duport-Dutertre.