Supporters of gay and lesbian rights marched to Mexico City's Metropolitan Cathedral in response to protests on Saturday opposing same-sex marriage.
Demonstrators carried banners saying "I respect your family, respect mine."
The cardinal of Mexico City, Norberto Rivera Carrera, denied the Catholic church was behind the protests in cities across the country.
In May, President Enrique Pena Nieto proposed a bill which would legalise same-sex marriage nationwide.
Gay marriage is legal in Mexico City and three other states. Some states have less formal arrangements.
The marches on Saturday were organised by a Catholic organisation, the National Front for the Family.
In a social media post, the Front said that around 300,000 people had taken part in 16 cities.
Local media reports put the numbers much lower.
Despite denial of involvement by the Catholic church, in some states priests were seen taking part.
Speaking to Reuters news agency, a member of the Front in the city of Queretaro said that the marches were to "awaken society in defence of the family, of natural marriage between a man and a woman".
There have not been such big marches over government legislation on social and religious issues in Mexico since the 1930s during the country's civil war, the BBC's Katy Watson in Mexico City reports.