The tournament had previously lost Australian Open champions Stan Wawrinka and Li Na but Williams’ 6-2 6-2 defeat by Spain’s Garbine Muguruza was on another level.
The world number one went into the tournament as a huge favourite to retain the title she won for the second time 12 months ago.
But, within an hour of her sister Venus losing to Anna Schmiedlova, Serena trudged off the court a beaten woman. The pair had been expected to meet in the third round.
The result was surprising enough but the manner of it added to the shock. The 32-year-old had never previously won fewer than five games in any grand slam match.
Meanwhile, coupled with Li’s loss to Kristina Mladenovic on Tuesday, it is the first time in the Open era that the top two seeds in the women’s tournament have lost before the third round at any grand slam.
Williams, who made 29 unforced errors and hit only eight winners, said: “I don’t think anything worked for me today.
“It was one of those days. You can’t be on every day, and, gosh, I hate to be off during a grand slam. It happens. It’s not the end of the world.
“I think she played really well and she played really smart. It’s great because I’m going to go home and work five times as hard to make sure I never lose again.”
As poor as Serena was, the match was just as much about the performance of 20-year-old Muguruza.
The world number 35 struck the ball with great power and precision and was nerveless serving out the biggest victory of her life.
The pair exchanged words at the net, with Muguruza explaining: “She said that if I continue playing like this, I can win the tournament. I said, ’I will try, I will try’.”
Venus performed much better against another young gun and raced through the first set but Schmiedlova showed impressive composure to turn the match around and win 2-6 6-3 6-4 to reach the third round at a grand slam for the first time.
Sharapova did not enjoy herself much in damp and gloomy conditions on Court Philippe Chatrier but recovered from 4-2 down in the opening set to defeat Tsvetana Pironkova 7-5 6-2.
Third seed Agnieszka Radwanska beat the rain and the darkness to defeat Karolina Pliskova 6-3 6-4 while there were also wins for Angelique Kerber, Dominika Cibulkova, Eugenie Bouchard, Carla Suarez Navarro, Sam Stosur and Daniela Hantuchova.
But Wimbledon finalist Sabine Lisicki had to pull out after hurting her wrist in a fall early on against
In the men’s singles, Novak Djokovic and Roger Federer showed the tennis world has not completely spun off its axis by moving smoothly into the third round.
Federer did not have things all his own way against qualifier Diego Sebastian Schwartzman but was comfortable enough in a 6-3 6-4 6-4 victory.
Djokovic, meanwhile, had the upper hand in every area of his match against Frenchman Jeremy Chardy as he eased to a 6-1 6-4 6-2 victory.
Djokovic faces a potentially tricky match next against 25th seed Marin Cilic, who beat Tobias Kamke in four sets, while Federer meets 31st seed Dmitry Tursunov.
There were also wins for French number one Jo-Wilfried Tsonga, sixth seed Tomas Berdych, eighth seed Milos Raonic, Gilles Simon, Tommy Robredo, Jerzy Janowicz and Ernests Gulbis.