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 successive weeks as world number one.
“Karolina played great today,” said Williams, who revealed she had been hampered by a sore left knee since earlier in the tournament
“I think if she had played any less then maybe I would have had a chance. So I think I wasn’t at 100%, but I also think she played well. She deserved to win today.”
Australian Open champion Kerber beat Denmark’s Carolina Wozniacki 6-4 6-3 to reach her first US Open final.
“It’s just incredible, it’s a great day,” said Kerber. “To be here in the final for the first time means a lot and to be number one in the world – it sounds amazing.”
Pliskova wins battle of big serves
Williams had won her only previous match against Pliskova two years ago
Pliskova went into the match on a 10-match winning streak, and with the most aces on the WTA Tour this year, and it was that shot which carried the Czech to victory.
It took Williams 50 minutes to earn her first break point as the Czech nullified her returns, while attacking the American’s second serve.
That brought Pliskova two breaks as she raced through the first set in just 26 minutes, and the home crowd on Arthur Ashe Stadium urged on Williams as the six-time champion fell behind midway through the second set.
Williams sparked into life, finally breaking Pliskova’s serve – to love – but the anticipated comeback was cut short in the tie-break.
There were a few signs of nerves as Pliskova let a 3-0 lead slip but from 5-4 in front, Williams produced a forehand wide, a backhand long and a double fault to end with 31 unforced errors.
“I don’t believe it,” said Pliskova, who had never made it past the third round of a Grand Slam in 17 prior attempts.
“I knew I had the chance to beat anyone if I played my game. I am excited to be in the final and to beat Serena as she is such a great champion.
“Even when she was down a set and break she was still fighting. I had to fight hard to win.”
Pliskova saved a match point against Venus Williams in round four and becomes only the fourth woman to beat both Williams sisters in the same Grand Slam tournament.
Russell Fuller, BBC tennis correspondent: “This was a nerveless and brilliant all-round performance by Pliskova, who – despite her success on tour – had not made it past the third round of a Grand Slam before this US Open.
“She has a big serve, and she used it, but also returned superbly throughout.
“The confidence gained from saving a match point to beat Venus Williams on the same court, in front of a similarly partisan crowd, in the fourth round helped Pliskova enormously.
“If she wins the final, there will have been four different Grand Slam champions in the women’s singles in 2016.”
Kerber follows in Graf’s footsteps
Kerber is through to her third Grand Slam final in 2016
Kerber, 28, was asked about her new-found number one status as she headed to court for the second semi-final, but said: “I’m just thinking about this match.”
That appeared the case as she raced into a 4-0 lead after just 16 minutes, her aggression from the baseline too much for Wozniacki, a former world number one now down at 74th in the rankings.
The Dane, 26, grabbed a foothold in the contest and there appeared to be a real edge between the two players, with Kerber complaining at length to the umpire about the noise emanating from Wozniacki’s player box.
Wozniacki saved two set points with backhand winners, but Kerber then served it out and powered into a 5-1 lead in the second that all but secured victory.
There was a final flurry of resistance from Wozniacki, who strung together nine points in a row to cut the deficit, before Kerber closed it out to become the 22nd WTA number one – and Germany’s second following Steffi Graf.
“For me, it’s just amazing to be, after Steffi, the next number one player in Germany,” said Kerber. “Steffi’s a great player and a great person, and I think she’s proud of me, to be the next one after her.”