Donald Trump vowed to “keep men out of women’s sports” during a crude, transphobic rant at a campaign rally in Alaska on Saturday night.
“It is actually demeaning to women, and it should not be allowed,” Mr Trump said.
Mr Trump criticised former University of Pennsylvania athlete Lia Thomas without naming her, claiming a “person with a man’s body” had unfairly won a women’s swimming competition.
In March, Ms Thomas became the first openly transgender athlete to win first place in the National Collegiate Athletic Association's (NCAA) first division swimming championship.
Her victory sparked a backlash against transgender athletes, and in June the world swimming’s governing body effectively banned transgender women from competing in women’s events.
Dozens of US states have introduced or are considering legislation to ban transgender student-athletes from playing on sports teams that align with their gender identity.
Mr Trump repeatedly misgendered Ms Thomas during the speech, which drew laughter from the crowd.
He also went on an extended rant about a transgender weightlifter who he claimed had broken records in a women’s competition. It’s unclear who he was referring to.
The former president has told variations of both stories at previous campaign rallies.
Mr Trump attended the rally in Anchorage to support candidates Kelly Tshibaka and Sarah Palin in their upcoming midterm races, while attacking incumbent Republican Lisa Murkowski, who voted to impeach him.