O'Donnell, the third of five children, was born and raised in Commack, Long Island, New York. She is the daughter of homemaker Roseann Teresa (nee Murtha) and Edward Joseph O'Donnell, an electrical engineer who worked in the defense industry. O'Donnell's father had immigrated from County Donegal, Ireland, during his childhood, and her mother was Irish American; O'Donnell was raised Roman Catholic. Her older brother is Daniel J. O'Donnell, now a member of the New York State Assembly.

In her January 31, 2002 appearance on the sitcom Will & Grace, she played a lesbian mom. A month later as part of her act at the Ovarian Cancer Research benefit at Caroline's Comedy Club O'Donnell came out as a lesbian, announcing "I'm a dyke!" "I don't know why people make such a big deal about the gay thing. ... People are confused, they're shocked like this is a big revelation to somebody." The announcement came two months before the end of the hosting of her talk show. Although she also cited the need to put a face to gays and lesbians, her primary reason was to bring attention to the gay adoption issue. O'Donnell is a foster and adoptive mother. She protested against adoption agencies, particularly in Florida, that refused adoptive rights to gay and lesbian parents. Diane Sawyer interviewed O'Donnell in a March 14, 2002, episode of PrimeTime Thursday. O'Donnell told USA Today that she chose to talk to Sawyer because she wanted an investigative piece on Florida's ban on gay adoption. She told Sawyer if that was done, "I would like to talk about my life and how (the case) pertains to me." She spoke about the two gay men in Florida who faced having a foster child they raised removed from their home. State law wouldn't let them adopt because Florida banned gay or bisexual people from adopting. O'Donnell's coming out drew criticism from some LGBT activists who cited her repeated references to being enamored of Tom Cruise on The Rosie O'Donnell Show as deceptive. She responded in her act stating, "I said I wanted him to mow my lawn and bring me a lemonade. I never said I wanted to blow him." After leaving her show and coming out, O'Donnell returned to stand-up comedy, and cut her hair. O'Donnell told the press that her haircut was meant to mimic the haircut of former Culture Club backup singer Helen Terry. O'Donnell was named 2002's "Person of the Year" by The Advocate, and in May 2003, she became a regular columnist for the magazine. The magazine's editor-in-chief, Judy Wieder, stated, "Today, Rosie's long and brave journey has led her not only to the cover of the Advocate - Rosie was honored with the magazine's Person of the Year Award for 2002 - but now to its chorus of voices, as a columnist."

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