[6] After he began modeling, Gubler had an internship with Wes Anderson, who encouraged him to audition for a part in his movie The Life Aquatic with Steve Zissou.

In 2014, Gubler acted in the zombie comedy Life After Beth and the film Suburban Gothic, for which he received the Screamfest Award for Best Actor.

He played Jimmy Olsen in All-Star Superman and voices the Riddler in the animated film, Batman: Assault on Arkham (2014).

While making The Life Aquatic with Steve Zissou, Gubler made a documentary titled Matthew Gray Gubler's Life Aquatic Intern Journal, a behind-the-scenes view of the making of the film, which was later included in The Criterion Collection DVD release.

[15] He directed and starred in a series of self-deprecating mockumentaries titled Matthew Gray Gubler: the Unauthorized Documentary[16][17] in which he parodies Hollywood behavior, which were filmed on the set of Criminal Minds.

[3][16][18] Gubler directed, edited, and co-produced the music video for "Don't Shoot Me Santa" by the rock band The Killers.

[12] In September 2005, the Gallery of Fine Art in Ostrava, Czech Republic, held a showing of 12 watercolors by Gubler; all the works were sold.

[21][22][23] Gubler contributed a painting of "Sandy The Mammoth" to the Western Heritage Museum & Lea County Cowboy Hall of Fame.

[28] Gubler wrote and illustrated his first book, Rumple Buttercup: A Story of Bananas, Belonging and Being Yourself, which was released on April 2, 2019.