Mathieu Amalric began his career in the film industry with a variety of experiences (prop boy, stage manager, canteen worker, etc.). Among other things, he was a trainee assistant on Louis Malle's Au revoir, les enfants (1997). He made his acting debut with Arnaud Desplechin and in 1997 won the César for Best New Male Actor for the film Comment je me suis disputé... (ma vie sexuelle). He went on to play a series of roles with André Téchiné (Alice et Martin, 1997), Olivier Assayas (Fin août, début septembre, 1998) and the Larrieu brothers (Un homme, un vrai, 2002).

He made several short films before directing his first feature in 1997, Mange ta soupe, followed in 2001 by Le stade de Wimbledon. In 2003, he directed La chose publique, presented at La Quinzaine des Réalisateurs.

At the same time, he continued his acting career. He reunited with Arnaud Desplechin for Rois et reines, which won him the César for Best Actor in 2005. He starred alongside Eric Bana and Matthieu Kassovitz in Steven Spielberg's Munich (2006), appeared in Sofia Coppola's Marie-Antoinette (2006) and played the lead in Julian Schnabel's Le Scaphandre et le Papillon, which won him a second César for Best Actor in 2008.

His third feature film, Tournée, won the Prix de la Mise en Scène at the 2010 Cannes Film Festival.