President Trump signed an executive order on Wednesday aimed at prohibiting transgender women and girls from competing in women’s sports, directing agencies to withdraw federal funding for any schools that refused to comply.

“From now on, women’s sports will be only for women,” he said in the East Room of the White House before signing the order.

The order, titled “Keeping Men Out of Women’s Sports” and signed on National Girls and Women in Sports Day, sought to deliver on an issue that Mr. Trump made a key theme of his campaign, which frequently denounced transgender athletes. Mr. Trump is relying on the Education Department to achieve the directive’s end through a revised interpretation of federal civil rights laws. Schools that do not follow these laws can lose federal funding.

The order also directed the State Department to push the International Olympic Committee to make similar changes at the international level by making eligibility “determined according to sex and not gender identity or testosterone reduction.”

The Trump administration is using the Education Department to carry out the policy by changing its interpretation of Title IX, the 1972 law prohibiting sex discrimination in programs that receive federal funding. The Biden administration had put forth a rule last year that made discrimination or harassment based on sexual orientation or gender identity a violation of federal civil rights law, but last month a federal judge vacated that regulation, providing Trump officials a path back to using the Title IX standards set in Mr. Trump’s first term.

In a statement after the order, the Education Department’s deputy general counsel, Candice Jackson, took up the president’s call to action, saying the department would “prioritize Title IX enforcement.”