Jankovic took the last five points to win in two hours 47 minutes
Jelena Jankovic saw off the second Williams sister in two days when she beat Serena 4-6 6-3 7-6 (7-5) to reach the final of the Italian Open.
Jankovic, who beat Venus on Thursday, saved two match points in the third set before Williams was broken both times.
Williams had a 5-2 lead in the tie-break but gifted Serbia's Jankovic the match with poor baseline shots.
Jankovic will face Maria Jose Martinez Sanchez, who beat Ana Ivanovic 6-4 6-2, in Saturday's final.
"To beat both Williams sisters in two days is really amazing," said Jankovic, who became only the seventh player to beat both Venus and Serena in the same tournament.
"I'm really proud of myself. It doesn't happen too often," added the 25-year-old, who joins Aranxta Sanchez-Vicario, Steffi Graf, Martina Hingis, Kim Clijsters, Lindsay Davenport and Justine Henin in achieving the feat.
Both women had started nervously, with world number one Williams the first to hold serve to take a 3-2 lead.
Williams took control with approach shots to the corners that allowed her to dominate at the net, and clinched the set with another break of serve thanks to a rasping forehand return that the Serb could only slice off court.
However, in the next set, an apparently tiring and increasingly error-prone Williams lost her serve to love as Jankovic, ranked seven in the world, went 4-3 in front.
And defending a set-point on her serve, the American double-faulted as the match went to a third set.
Both players managed three breaks apeice in the final set but Jankovic had gained a psychological advantage going into the tie-breaker after saving two match points.
It looked ominous for Jankovic, though, when Williams took a 5-2 lead in the decider.
Martinez Sanchez has two WTA singles titles to her name
An unforced error made it 5-3 before the Serb thought she had aced on the next point, but the chair umpire ruled that Williams, who had her hand up, had called for Jankovic to delay her serve.
Jankovic's protestations fell on deaf ears and drew whistles from the crowd at the half-full stadium in Rome.
"For me, when the server comes to the line the receiver had to be ready. That's the rule, right?" Jankovic reflected afterwards.
"I just had to regroup and refocus. I didn't want to waste my energy - and that's what I basically did."
In truth, Jankovic's final steps to victory had more to do with the 12-time Grand Slam champion's lack of concentration than her own mental fortitude.
Williams, though, was not in the mood to dwell on her profligacy having been out with a knee injury since winning the Australian Open in January.
"I wasn't playing the points right. I feel like I should have won, could have won," she said.
"I think I had an apple in my throat, but it is what it is. I can't beat myself up over it - I just came back."
Spaniard Martinez Sanchez stands in the way of Jankovic's third title at Foro Italico, after the 26th-ranked Spaniard's serve-and-volley game proved too strong for former world number one Ivanovic - now ranked 58.
In March, Jankovic beat Caroline Wozniacki to win the BNP Paribas Open at Indian Wells.