Czech 10th seed Karolina Pliskova outplayed Serena Williams to reach the US Open final and end the American's reign as world number one.
Williams, 34, double faulted on match point to lose 6-2 7-6 (7-5), and Pliskova will face German second seed Angelique Kerber in the final.
The result guarantees that Kerber will overtake Williams at the top of the rankings after the US Open.
Williams had also hoped to win an Open-era record 23rd major singles title.
She remains tied with Steffi Graf for both Grand Slam titles, and the WTA record of 186 weeks as world number one.