The small group, which reportedly only consisted of about 10 people, are believed to have carried signs with slogans such as “trans activists erase lesbians” and distributed leaflets stating “a male can never be lesbian”.
Tens of thousands of people battled temperatures of 30C to join the parade this afternoon.
But after it was officially opened by London mayor Sadiq Khan, a small group of activists moved to the front of the parade holding anti-trans banners and distributing leaflets.
Pride in London is facing a backlash for not moving the protesters on sooner.
But organisers said they “absolutely don’t condone” the messages distributed by the anti-transgender activists and said they directed the group away from the parade as quickly as possible.
A Pride in London spokesperson told HuffPost UK that the small group appeared as photos were being taken and, due to the hot temperatures, the parade was allowed to move forward before the protesters were “directed away from the parade”.
The spokesperson added: “Every year, Pride is attended by hundreds of thousands of people who demonstrate that Pride still matters. Given the hot weather and in the interest of the safety for everyone attending today’s event, the parade group was allowed to move ahead.
“We do not condone their approach and message and hope the actions of a very small number people does not overshadow the messages of the 30,000 people marching today.”
A copy of the leaflet reportedly circulated by the anti-trans activists was shared on social media.
Pride in London was criticised for not taking a firmer stance at the time of the incident, with the LGBT+ and Liberal Democrat group calling on organisers to apologise and resign.
The group’s chair, Jennie Rigg, said: “I am appalled that transphobic protestors were allowed to lead the march and the crowd asked to cheer them on. This is a betrayal of the thousands marching. The Pride organisers should resign and offer a full apology.
“Allowing them to continue betrays the LGBT+ communities by sowing division and hatred at an event aimed at fostering inclusion, love and pride.
“Lib Dems are committed to preserving and extending rights for trans people.”
The capital was awash with rainbows, glitter and love at this year’s parade.
Pride director Christopher Joell-Deshields said this year’s parade was the most diverse, but Stonewall pulled out of this year’s festival saying it had concerns about the event’s “lack of diversity”.
The charity said it will instead extend support to UK Black Pride, an event for LGBT people from ethnic minorities that also takes place every year in the capital.
About 30,000 people from 472 organisations took part in the march through the capital’s centre.
There were shouts and cheers as the parade got under way with a huge rainbow flag in Regent Street.
The streets were full of balloons, sequins and flags as thousands of supporters lined the route.
The event also coincides with the 70th anniversary of the NHS and a banner supporting the organisation took pride of place at the front of the march.