Having trailed by a set and a break, Williams looked to have powered her way out of trouble when she moved 5-1 ahead in the deciding set.
But, incredibly, Pliskova won six games in a row to set up a last-four clash with Naomi Osaka, saving one match point at 5-1 and three more at 5-4.
The Czech was rewarded handsomely for her bravery on the big points while Williams appeared to struggle with her movement after jarring her left ankle on the first match point.
Pliskova, who is in the running to finish the tournament as world number one, said: "I was almost in the locker room but now I'm standing here as a winner so it's a very good feeling. My mind was in the locker room. It was tough because she was really playing well.
"I was a little bit passive, mentally down. I just said at 5-2 maybe I'm going to have a chance. She got a little bit shaky, I took my chances and I won."
After Osaka's victory over Elina Svitolina, the script appeared to be written for a rematch of her controversial US Open final victory over Williams.
The American has looked fitter and sharper here than she did last season, with coach Patrick Mouratoglou saying she was mentally and emotionally ready to win a 24th grand slam singles title, finally equalling Margaret Court's record.
Williams lost to Pliskova at the US Open in 2016, with the 26-year-old reaching her only slam final, but beat her in New York last year having made a sluggish start, a pattern that was repeated here.
Williams trailed 3-1, saved four break points to avoid going 4-1 behind but this time could not find a response in the opening set, and the 16th seed looked in trouble when she was broken for 3-2 in the second.
However, Williams at last got her feet moving and responded straight away before stepping up her aggression to level the match.
All appeared to be going smoothly in the third set for Williams as she moved 5-1 ahead and held a match point on her own serve but, after being called for a foot fault, she jarred her left ankle and was broken.
She still had a good cushion but, when Pliskova won a third straight game to get back on serve, it seemed the unthinkable comeback might be on.
The seventh seed then passed a huge test at 5-4, trusting her big forehand and saving three more match points.
Suddenly the momentum was all with Pliskova and, although Williams saved two match points herself, a netted forehand on the third brought the most dramatic of afternoons to a wholly unexpected conclusion.