The pop star sang a few bars of her song "Born This Way" but spent most of her time on stage speaking against intolerance and discrimination against the gay and transgender communities.
"It has become clear to me that my greater mission is to be part of the joyous mobilization of the LGBT community worldwide," she said to a cheering crowd.
Lady Gaga told the crowd she is often asked "How gay are you, Lady Gaga?"
"My answer is: 'I am a child of diversity."'
Her appearance came after the weekend-long European gay pride celebrations.
Organizers said they hope the event will draw attention to discrimination gays face in many parts of the world. Organizers also said Rome was a significant choice of venue, since it is home to the Vatican, which staunchly opposes legislation that would recognize same-sex marriage or adoption by gay couples.
The Associated Press contributed to this report.