Pliskova beat both Williams sisters on her way to the U.S. Open final.
For Serena Williams, the wait for Grand Slam title No. 23 will have to wait until next year.
Williams was upset in the U.S. Open semifinals on Thursday night by Karolina Pliskova, who earned a 6-2, 7-6(5) victory at Arthur Ashe Stadium.
Pliskova, the No. 10 seed, hadn't advanced past the third round of a Slam prior to this year's U.S. Open, where the Czech Republic native has earned victories over both Williams sisters en route to the final.
Williams's defeat also means that she will lose her No. 1 ranking, which she has held for 186 consecutive weeks. Angelique Kerber will take over as the top–ranked player in the WTA.
The last player to beat both Venus and Serena Williams in a Slam was Kim Clijsters at the 2009 U.S. Open. Martina Hingis and Justine Henin are the only other players to take down the Williams sisters at a Slam.
"Probably right now America hates me because I beat both sisters Williams," Pliskova said on ESPN after the match.
Williams has 22 Grand Slam titles, tied with Steffi Graf for the most in the Open Era. Williams said after the match that while she wasn't fatigued from Wednesday's quarterfinal, she struggled with movement because of a knee issue.
"I have been having some seirous left knee problems," Williams said. "I wasn't tired. Fatigue had absolutely nothing to do with it."
Pliskova, 24, will face either Kerber in Saturday's final.