Gay, lesbian and transgender activists and sympathizers, dancing on floats to electronic music and waving rainbow flags, held up signs reading "In France I can now get married, when in Italy?"
"People's rights cannot be negotiated. Rome will become the capital of rights. There have been too many attacks in the last few years, too many tragedies of loneliness," the city's new centre-left mayor Ignazio Marino said.
Tanned men in sailor outfits, drag queens in jewelled headdresses, and women with nipple tassels and painted buttocks swanned past the Colosseum, many brandishing banners saying "Close down the Vatican! Guantanamo mentality".
Italy does not recognize gay and lesbian marriage or same-sex civil unions and rights campaigners have blamed the influence of the Catholic Church for hampering legislation.
At the national Pride day in Palermo on Friday, Italy's new equal opportunities minister Josefa Idem promised "a bill on civil unions, something increasing numbers of citizens are rightly asking for".
The first minister to attend Gay Pride in Italy, Idem said she had come "tobring a concrete signal of the government's attention to the requests from society which cannot be silenced".
Idem said she would fight for an amendment to Italy's hate crimes law to include homophobic offences in a bid to tackle gay discrimination.
Laura Boldrini, the president of the lower house of parliament who also attended the Palermo Pride, said victims of homophobia must be protected because "homophobes, chauvinists and racists are all children of the same subculture fed by prejudice".
At the end of May, a lawmaker from Silvio Berlusconi's People of Freedomparty said he was submitting a draft bill to recognise same-sex unions following France's first gay weddings.
France celebrated its first official gay marriage at the end of May after months of sometimes violent protests.
Giancarlo Galan said the proposed Italian law would apply the "same rules" as marriage, giving gay couples "the same rights and duties as heterosexual ones," particularly on inheritance and pensions.
Gay rights organisations voiced approval but pointed out that previous draft bills on the subject have never been approved.