The 65-year-old announced her upcoming departure during a talk at the American Library Association Annual Conference earlier this week.
Manzano, who starred alongside TV husband and Fix-It Shop co-owner Luis, played by Emilio Delgado, since 1971, said she would not return in Sesame Street's upcoming 46th season.
Raised in a poor Puerto Rican family in New York's tough Bronx neighbourhood, Manzano attended the High School of Performing Arts, which was the setting of the 1980 film Fame.
She was cast in the role of Maria shortly after graduating from Pittsburgh's Carnegie-Mellon University.
In a video shared Tuesday by the American Library Association, Manzano said Seasame Street's early target audience was children in America's inner cities, and having a multicultural cast was part of that goal.
"The stoop in Harlem was the most familiar to [inner city kids]," Manzano explains. "And of course, populating the stoop were people of colour and Latin people and Jewish people ... so that these kids could find someone on TV that they could relate to."
Manzano didn't just perform on Sesame Street. She also wrote for the show that teaches numbers, colours and the alphabet using a cast of Jim Henson's Muppets.
Manzano has won 15 Emmy Awards as part of thewriting staff.
Sesame Street fans react
News of her departure triggered an outpouring of emotion from fans who grew up with her as Maria.
While PBS hasn't formally announced Manzano's retirement, the actress has been actively responding to fans on Twitter.
She even hinted that she might return for the show's 50th anniversary, in November 2019.