President Donald Trump signed an executive order Wednesday banning transgender athletes from participating in women's sports, fulfilling a promise that was at the center of his 2024 campaign.

The order will establish sweeping mandates on sex and sports policy and will direct federal agencies, including the Department of Justice, to interpret federal Title IX rules as prohibiting the participation of transgender girls and women in female sports categories, according to a White House document on the executive order obtained by ABC News.

Titled "Keeping Men Out of Women's Sports," the order will mandate immediate enforcement, including against schools and athletic associations that "deny women single-sex sports and single-sex locker rooms," according to the document, and will direct state attorneys general to identify best practices for enforcing the mandate.

Surrounded by female athletes at the signing ceremony, Trump railed against what he called "transgender lunacy."

"Under the Trump administration, 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 said.

He also falsely claimed that Algerian boxer Imane Khelif -- who faced attacks over her sex and gender during the 2024 Paris Olympics -- was "a male," despite identifying a woman and having been assigned female at birth, according to the IOC.

The White House expects sports bodies like the NCAA to change their rules in accordance with the order after it is signed, according to a senior administration official.

"We're a national governing body and we follow federal law," NCAA President Charlie Baker told Republican senators at a hearing in December. "Clarity on this issue at the federal level would be very helpful."

Despite right-wing ire, there are very few transgender athletes in women's sports. In December, Baker said in Senate testimony that out of 500,000 NCAA athletes, he is currently aware of fewer than 10 that are trans.

Trump was joined at the ceremony by athletes, coaches and advocates who have campaigned against transgender participation in women's sports. More than 60 attendees, including former University of Kentucky swimmer Riley Gaines, joined the ceremony.

"We want to take actions to affirmatively protect women's sports," deputy assistant to the president and senior policy strategist May Mailman told ABC News, adding that the executive order is designed to further overturn Biden-era policies that required schools and athletic organizations to treat gender identity and sex as equivalent. She noted that a court ruling determined such requirements were not necessary, and that the president's executive order would explicitly ban them.

Trump's executive order will lead to increased discrimination and harassment, Human Rights Campaign President Kelley Robinson said in a statement on Tuesday.

"This order could expose young people to harassment and discrimination, emboldening people to question the gender of kids who don't fit a narrow view of how they're supposed to dress or look," Robinson said. "Participating in sports is about learning the values of teamwork, dedication, and perseverance. And for so many students, sports are about finding somewhere to belong. We should want that for all kids – not partisan policies that make life harder for them."

Mailman said the executive order's goal was "not to make sure that everybody conforms to their sex stereotype as they're playing sports" but to "protect women's sports," adding that options like co-ed categories would still be available.

If universities don't comply, the White House warned they could not only lose federal funding but also face legal action.

"If schools don't comply, it's not just that they're at risk of DOJ-based actions," Mailman said. "Title IX has a private right of action component behind it, so if schools are violating the law, they're at risk of lawsuits from their female students, that is going to actually be more than just taking away federal funding. These are multi-million dollar lawsuits."

The Daily Wire was first to report that Trump would sign the executive order on Wednesday.

The executive order also directs the secretary of state to push for changes within the International Olympic Committee to maintain single-sex competition and the Department of Homeland Security to review visa policies to prevent transgender women from identifying as female, which would allow them to compete in women's sports, according to the document detailing the order.

The order will also direct private sporting bodies to attend an in-person White House meeting to hear accounts from female athletes in person, according to the document.

"The Executive Order calls for the convening of private sporting bodies in the White House to hear, in person, the stories of female athletes who suffer life-long injuries, who have been silenced and forced to shower with men, and whose hard work has been cast aside due to the biological advantage of males," the document states.

The order is the most aggressive move yet by Trump to fulfill one of his central campaign promises regarding transgender athletes in women's sports.

Trump signed an executive order last week seeking to restrict gender-affirming care for people under the age of 19.

The order would move to restrict medical institutions that receive federal funding from providing such care -- including puberty blockers, hormone therapies, and surgeries -- calling on the secretary of the Department of Health and Human Services to "take all appropriate actions to end the chemical and surgical mutilation of children."