Poehler's publicist confirmed to Reuters that the couple had decided to separate.
The pair, who often appeared together on screen, have been married for nine years and have two young sons.
"It's very amicable," an unnamed source told Us Weekly.
Poehler, 40, is the star of TV comedy "Parks and Recreation" and became a household name performing on "Saturday Night Live."
Arnett, 42, is a Toronto native known for his roles on TV series including "Arrested Development" and "Up All Night."
(Reporting By Nichola Groom, Editing by Jill Serjeant and Eric Walsh)