The media mogul will star opposite Tony-winner Audra McDonald for the play.
Winfrey, 60, will portray the role of a mother struggling to stop her daughter from killing herself, reported the New York Times.
Tony winner George C Wolfe, who recently directed Tom Hanks in Nora Ephron's Lucky Guy, would direct the production, which is being eyed for the 2015-16 Broadway season.
The play is being produced by Scott Sanders.
Winfrey has shown more keenness in acting assignments in recent times.
She is also teaming up with Sanders for a Broadway revival of her hit film 'The Color Purple' with a possible 2014-15 season.
'night, Mother' was written by Marsha Norman, who previously teamed up with Winfrey and Sanders on the book for 'The Color Purple' musical.