An extraordinary decider saw Pliskova, chasing a maiden Grand Slam, win six games in a row to seal a 6-4 4-6 7-5 victory and end Williams’ hopes of a record-equalling 24th major.
The standout moment – and arguably the turning point – of the final set saw Williams called for a foot fault on her first match point, before going on to injure her ankle in the rally that followed as Pliskova eventually broke back to make it 5-2.
“My mind was in the locker room but I was still here,” said Pliskova. “She was playing really well, I was too passive, but she got a little bit shaky and I took my chances.”
In reaching the last four for the first time in Melbourne, Pliskova ensured any rematch between Williams and Naomi Osaka – four months after their highly-charged US Open final meeting – would have to wait.
Williams led her head to head with Pliskova 2-1 having won their previous encounter at the same stage of last year’s US Open.
Seeking revenge on the Rod Laver Arena, Pliskova broke three games in, and then squandered four opportunities to take a 4-1 lead as Williams dug deep to hold.
Pliskova continued to assert her dominance, however, and saw out the set after 40 minutes without offering the American a single break-back opportunity.
Williams saved another break point in the opening game of the second set before holding, but she was powerless to prevent Pliskova from breaking in the fifth.
In true champion style, Williams broke back to love, and in a lengthy 10th game she converted her first set point to send the match to a decider.
It was seemingly one-way traffic in the final set, with Williams effortlessly breaking twice to move 5-1 in front.
But when looking to serve it out, a frantic first match point followed, with Williams penalised for a foot fault before appearing to roll her ankle as Pliskova won the point in the subsequent rally. At deuce, a double fault then shot into the net from Williams allowed Pliskova to secure the first of two breaks required to stay in the match.
Pliskova held her next service game before breaking again, this time to love, with Williams visibly struggling after hurting her left ankle.
Williams appeared to shrug off the ankle problem in the next game, but failed to convert three more match points as Pliskova rallied to level at 5-5.
An enthralling finale then saw Pliskova break to love once more, and though Williams saved two match points of her own, the Czech sealed a stunning victory at the third time of asking.
“It’s definitely not easy for me. From day one, I expect to go out and, quite frankly, to win,” the seven-times champion in Melbourne told reporters.
“That hasn’t happened. But I do like my attitude… I’ve only been playing 10 months, but I expect to win, and if I don’t, it’s disappointing.
“I rather think of it that way and know that it’s going to happen sooner or later than making an excuse for myself. I don’t like making excuses.”