Gunn and his brothers all attended the Jesuit St. Louis University High School, where he graduated in 1984. He went on to earn a Bachelor of Arts from Saint Louis University. While at Saint Louis University, Gunn created political cartoons for the school's student weekly, The University News. Gunn said that, at an unspecified time in his college education, "I went to two years undergraduate film school at Loyola Marymount in Los Angeles. But I was pretty screwed up at the time, and had to leave. Years later I went to graduate school at the Columbia University School of Fine Arts but I studied prose writing, not film writing." He earned a Master of Fine Arts from Columbia University in 1995.

While living in St. Louis, Gunn started a band, The Icons, in 1989, serving as lead vocalist. The group released the album Mom, We Like It Here on Earth in 1994, and its songs "Sunday" and "Walking Naked" were featured in the film Tromeo and Juliet. The Icons disbanded in the mid-1990s. Gunn has continued to work in music, composing songs for Scooby-Doo, Scooby-Doo 2: Monsters Unleashed, and Movie 43.

Gunn began his career in filmmaking with Troma Entertainment in 1995, for which he wrote the independent film Tromeo and Juliet. Working alongside his mentor Lloyd Kaufman, the co-founder of Troma, Gunn learned how to write screenplays, produce films, scout locations, direct actors, distribute films, and create his own poster art. After contributing to several other Troma films, Gunn in 2000 wrote, produced and performed in the superhero comedy The Specials, directed by Craig Mazin and featuring Rob Lowe, Thomas Haden Church, Paget Brewster, Judy Greer and Jamie Kennedy.

Gunn married Actress Jenna Fischer on October 7, 2000, in an event that Gunn's hometown paper said was officiated by Lloyd Kaufman, but which Gunn later clarified had not been: “They said I was married by Lloyd Kaufman, which is not true. He was at my wedding and he gave a speech.” The two had originally met in the St. Louis area through Gunn's brother Sean, who had acted in plays alongside Fischer in high school. After seven years of marriage, Gunn and Fischer announced their separation in a joint statement on September 5, 2007, divorcing in 2008. In 2010 Fischer helped Gunn with casting Rainn Wilson, her co-star on The Office, in Gunn's film Super.

Gunn's first major Hollywood screenplay was Scooby-Doo in 2002. In 2004, he wrote the screenplays for the remake of Dawn of the Dead and the sequel Scooby-Doo 2: Monsters Unleashed. With these films, Gunn became the first Screenwriter to have two films top the box office in consecutive weeks. That same year, he executive produced and starred in the mockumentary LolliLove, directed by and starring his then-wife Jenna Fischer. His film directorial debut was the 2006 horror-comedy Slither, which was included on Rotten Tomatoes' list of the 50 Best Ever Reviewed Horror Movies.

In 2008, Gunn was a judge on the VH1 reality television show, Scream Queens where 10 unknown actresses compete for a role in the film Saw VI, where he directed contestants during acting challenges.

In 2009, Gunn announced he was going to write and direct Pets, a comedy about a man who is abducted by aliens who want to turn him into a household pet, with Ben Stiller, Stuart Cornfeld and Jeremy Kramer producing. However, by March 2009, Gunn announced, "Pets unfortunately, is done. I'm gone. I left the project for various reasons. I hope it sees the light of day somehow, but it won't be with me attached as Director."

In 2010, Gunn released the film Super, a dark comedy and superhero satire starring Rainn Wilson and Ellen Page.

Growing up, Gunn was influenced by low-budget films such as Night of the Living Dead and Friday the 13th. He read magazines like Fangoria and attended genre movie screenings, including the original Dawn of the Dead at the Tivoli Theatre in St. Louis. At the age of 12, he began making 8 mm zombie films with his brothers in the woods near their home.

Gunn co-wrote and directed the Marvel Studios adaptation of Guardians of the Galaxy, which was released on August 1, 2014. His brother Sean Gunn has a role in the film. Gunn has appeared as an actor, mostly in smaller roles or uncredited appearances in his own projects.

Gunn wrote and directed Guardians of the Galaxy Vol. 2 (2017). In April 2017, Gunn confirmed he will return to write and direct Guardians of the Galaxy Vol. 3.