"From now on women's sports will be only for women," Trump said before he signed the order at the White House, surrounded by dozens of children and female athletes.

"With this executive order the war on women's sports is over."  

Top Republicans including US House Speaker Mike Johnson and firebrand Congresswoman Marjorie Green were among those in the audience to watch the signing ceremony.

"We will defend the proud tradition of female athletes, and we will not allow men to beat up, injure and cheat our women and our girls," Trump added to applause and cheers.

Trump's order allows US government agencies to deny funds to schools that allow transgender athletes to compete on women's teams.

"If you let men take over women's sports teams or invade your locker rooms, you will be investigated... and risk your federal funding," Trump said.

Trump said he would now push the International Olympic Committee (IOC) to change its rules on transgender athletes before the 2028 Los Angeles games.

He said he had ordered Secretary of State Marco Rubio to "make it clear" to the IOC that "we want them to change everything having to do with the Olympics and having to do with this absolutely ridiculous subject."

Trump added that he had directed Homeland Security chief Kristi Noem to deny visa applications to "made by men attempting to fraudulently enter the United States while identifying themselves as women athletes to try and get into the Games."

Republican Trump has repeatedly targeted gender non-conforming people in a blitz of orders pushing his radical right-wing agenda at the start of his second term.

At his inauguration speech Trump announced that US government policy would only recognize two genders, male and female -- ending the practice of providing a third gender option in some settings.

Days later Trump signed an order to rid the military of what he called "transgender ideology" and effectively ban transgender troops. He also issued an order to restrict gender transition procedures for people under age 19.

The slew of actions comes despite the fact that transgender people make up only a small minority of the US population.

During the 2024 election campaign he repeatedly hammered Democrats on the divisive issue of transgender rights during the 2024 election campaign, capitalizing on a broader culture war over the topic.

One of Trump's most successful attack lines against his election rival Kamala Harris -- "Kamala Harris is for they/them. President Trump his for you" -- targeted her support for trans rights.

Trump also targeted her one-time support for gender reassignment operations for prisoners.

His executive order on Wednesday comes after the Republican-led House of Representatives in January passed a bill severely restricting transgender athletes from participating in girls' and women's sports.

As transgender people have become a more visible presence in the United States, sparking questions around gender norms and fairness, many conservatives have rallied around women's sports.