The Curb Your Enthusiasm creator and actor - who was first linked to the project in February - will take on the role of Mother Mengele in the Farrelly brothers' film based on the classic TV shorts, reports Deadline.
The character is in charge of running the orphanage where the Stooges grow up and "learn to create mayhem".
Jane Lynch was previously confirmed to play Mother Superior, an enemy to the Stooges who attempts to have them thrown out of the orphanage.
Will Sasso previously signed on in the role of Curly while Will and Grace actor Sean Hayes will take on Larry in the film.
Mike Cerrone, who previously collaborated with the Farrelly brothers on Me, Myself and Irene, has joined the siblings to write the screenplay.
Shooting on The Three Stooges is expected to begin later this month.