Karolina Pliskova beat the 37-year-old Williams 6-4 4-6 7-5 in a shade past two hours, the second time the Czech has toppled the legendary American at a major, after the 2016 US Open.
During that event, the former world No. 1 downed both Williams and older sister Venus.
Williams led 5-1 in the third set Wednesday but the complexion changed completely when she turned her ankle on her first match point in that seventh game changing direction.
She immediately double faulted and her serve altogether waned, Williams losing all 10 points on her serve the rest of the contest.
Pliskova saved three more match points at 4-5, one with a sizzling forehand winner.
Williams had looked sharp throughout this fortnight, too, breezing through three rounds before beating current world No. 1 Simona Halep in a high-quality, three-set tussle Monday.
Rare quarterfinal loss
Losing in the last eight at a major is exceedingly rare for Williams — she had been 14-1 at this stage in her last 15 grand slam appearances.
But her last loss came here in Melbourne in 2013, ousted that day too as injuries played a part against Sloane Stephens.
Overall it was Williams’ earliest departure at a grand slam — not including a withdrawal — since a third-round showing at Wimbledon in 2014.
Returning to action last year after giving birth to daughter Alexis Olympia in September 2017, Williams impressively made finals at Wimbledon and the US Open, falling to Angelique Kerber and Osaka, respectively.
When Williams and Osaka met in the final in New York in September, it turned out to be one of the most memorable grand slam finals in recent history.
And not for the forehands and backhands on display.
Williams accused chair umpire Carlos Ramos of sexism and called him a “thief” in her straight-set loss.
That controversy overshadowed Osaka’s terrific play, but the Japanese star has admirably kept it going.
The 21-year-old beat Elina Svitolina 6-4 6-1 earlier Wednesday to became the first female player since Kim Clijsters in 2006 to reach a grand slam semifinal after winning her maiden major at the previous grand slam.
Pliskova to kick on?
Is Pliskova on course for a maiden major herself after her dramatic comeback?
The 26-year-old is ever dangerous and some might say has underachieved at grand slams because of her massive game. Like Williams, her sister plays tennis, in this case twin sister Krystyna.
Perennially one of the biggest servers in tennis, Pliskova found all her serving spots in the early going.
Indeed, Williams didn’t manufacture a break point until the second set, by which time Pliskova grabbed a set lead.
That didn’t bode well for Williams, since she was 2-6 on her comeback when losing the first set.
Pliskova — with a new coaching team that includes former Wimbledon champion Conchita Martinez — broke Williams for 2-1 in the first. When she squandered four break points in the fifth game, it felt it could be a turning point.
Pliskova however refused to buckle and got some help trying to serve out the opener when Williams erred on a drive volley on the first point with her foe completely stranded.
A relieved Pliskova went on to hold.
Pliskova initially blinked
Close to pulling off the upset at 3-2 in the second — yes an upset, no matter what the current rankings suggest — Pliskova blinked and was broken to love for 3-3.
Williams had the momentum and pounced in the final game of the set, breaking after Pliskova led 40-15. In that game, Pliskova failed to take any of her five game points.
A break in the fourth game of the third seemingly set Williams on her way prior to the incredible proceedings on that first match point.
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