The Parks and Recreation actress and the Up All Night actor married in 2003 and have two sons together, 3-year-old Archie and 2-year-old Abel.
The couple's rep confirmed the news to Us Weekly, with a source adding: "It's very amicable."
Poehler and Arnett have frequently starred in the same projects together, including the films Blades of Glory, Spring Breakdown and Monsters vs. Aliens.
Arnett is currently working on the fourth season of Arrested Development, which began filming last month and will air on Netflix in 2013.
Poehler's Parks and Recreation returns to NBC with a 22-episode fifth season on Thursday, September 20.
Watch Amy Poehler and Will Arnett in a deleted scene from Parks and Recreation below: