Rust was born in Le Mars, Iowa, the son of Jeanne and Bob Rust. He was raised Catholic and attended Gehlen Catholic High School. His mother was a Teacher at Le Mars Community High School, and his father owned a Western goods and boot-repair shop. He graduated from the University of Iowa in May 2004.
Rust is best known as a stand-up and Sketch Comedian at the Upright Citizens Brigade Theater (UCB) in Los Angeles. At UCB, he is currently a member of the Sketch comedy groups A Kiss From Daddy and Last Day of School. He has appeared in films such as Inglourious Basterds, Ass Backwards, Freak Dance, Paper Heart and Semi-Pro. He made his most high-profile acting role in the summer of 2009, playing the male lead in the film I Love You, Beth Cooper. Rust has written for television programs such as Human Giant and Moral Orel. Rust and Comedian Charlyne Yi formed the band The Glass Beef in 2006. The two appeared together in a video for "Song Away" by Hockey. He is also the lead singer and bass player in the comedy rock trio Don't Stop or We'll Die, with fellow comedians Michael Cassady and the late Harris Wittels.
In November 2010, Rust appeared in the Comedy Central Sketch comedy special This Show Will Get You High, created by and starring Matt Besser. He and Paul Reubens co-wrote the Pee-Wee Herman film Pee-wee's Big Holiday, which was released in 2016.
Rust married Writer Lesley Arfin in October 2015. Rust said in one interview that Arfin fell in love with him when she learned that he had defecated (intentionally) on stage during a comedy show. In October 2017, they welcomed a daughter, Mary James.
Rust stars in the Judd Apatow-produced Netflix series Love, which premiered in February 2016. Rust co-created the show with his wife Lesley Arfin.
