Following the death of his father he became one of the leaders of the Catholic faction in the French Wars of Religion, and he was prominent in the St. Bartholomew's Day Massacre in 1572.
In 1584 he formed a league to keep the new heir, the protestant Henry of Navarre, off the throne. This led to the stage of the Wars of Religion known as the War of the Three Henries. Henry III failed to meet the demands of the Catholic League, and on May 12, 1588 Guise entered Paris, forcing Henry to flee.
Henry then agreed to all Guise's demands, but later had him assassinated by the his bodyguard at Blois.