Stosur, who had never beaten the older Williams sister before, booked her place in the semi-finals of the first clay-court tournament of the season with a rallying win over Venus that took two hours and 18 minutes.
The Australian was forced to return to court on Friday morning to complete her rain-delayed fourth-round clash against Galina Voskoboeva, in which she triumphed 6-3 4-6 6-2, and was obviously tired in the middle stages against Venus.
Stosur raced through first set, quickly taking a 5-2 lead before serving out at the first attempt when Venus mis-hit a return.
But the reigning US champion’s serve deserted her in the second set and the American, playing just her second tournament since withdrawing in New York last September after receiving a diagnosis or Sjogren’s Syndrome, bounced back to level the clash.
But as quickly as it had disappeared, Stosur regained her accuracy on serve and, from 2-1 down in the final set, won five of the last six games to set up a last four clash against Serena Williams.
Stosur beat Serena in last year’s US Open final, as well as emerging the victor in their only other clash – at the French Open quarter-finals in 2010.
"For sure, clay's not my favorite surface," said Stosur, the champion in Charleston in 2010. "It's always tough playing Serena no matter where."
Venus, handed a wildcard to play at Key Biscayne in Miami last week, will rise another 17 ranking places when the new rankings are announced on Monday.
"I've never been so happy to be 70th" in the world, Venus said.
"I did my best. I know I can play even better. My errors didn't help my cause. At this point, I'm just learning and learning and learning more.
"I have come so far, so far from the US Open.”
Serena booked her place in the last four when Germany’s sixth seed Sabine Lisicki was forced to retire with a twisted ankle.
Lisicki, who missed five months in 2010 with an left ankle injury, looked in the early stages like she might cause Serena some real trouble; the German going toe-to-toe with the 13-times Grand Slam champion from the baseline.
But, in just the second game, the German collapsed to the court as she moved to her left for a shot. Lisicki immediately called for the trainer, who strapped her left ankle in order that she could continue.
It was clear that the German could not put much pressure on the left leg, however, and three games later, while trailing 4-1, a tearful Lisicki called it quits.
"We'll see what the doctors say and we'll go from there," she said.
Lisicki will have an MRI scan on the injured ankle.
Fourth seed Vera Zvonareva also departed the tournament, beaten 6-3 6-3 by Lucie Safarova. The ninth-seeded Czech will face 14th seed Polona Hercog in the last four, after the Slovenian blitzed 13th-seeded Nadia Petrova 6-1 6-2.
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