Less than a month after winning the French Open, the second-seeded Spaniard was beaten in the second round of Wimbledon on Thursday by a Slovakian qualifier ranked No. 124.
In the biggest upset of the tournament so far, Jana Cepelova beat a listless Muguruza 6-3, 6-2 on Court 1 in less than an hour to reach the third round at the All England Club.
Muguruza looked like a shadow of the player that reached the final last year, where she lost to Serena Williams, and beat Williams in the final at Roland Garros this month for her first major championship.
Muguruza sprayed ugly shots all over the court Thursday and finished with 22 unforced errors, compared to just nine winners. Cepelova had a first-serve percentage of just over 40 percent, but still outplayed Muguruza with 14 winners and 12 errors.
"I played really well, and I think I deserved the victory," Cepelova said.
Cepelova, Slovakia's No. 5 female player, had to win three matches in qualifying to make it into the main draw. It's not the first time she's knocked off a top player: Cepelova ousted Simona Halep in the first round at Wimbledon last year, also on Court 1, and beat Serena Williams at a tournament in Charleston in 2014.
"Last year I beat Halep on the same court and I had great memories," the 23-year-old Cepelova said. "I am so happy."
Earlier, five-time champion Venus Williams led a group of Americans picking up victories as the tournament scrambled to get back on schedule after two days of bad weather.
With play starting earlier on the outside courts and the rain holding off, Williams was among nine American players winning first- or second-round matches.
U.S. men's winners included John Isner, Sam Querrey, Donald Young, Jack Sock and Steve Johnson. Joining Williams among the women's winners were Sloane Stephens, Madison Keys and Julia Boserup.
The 36-year-old Williams, the oldest player in the women's draw, needed nearly 2½ hours to get past 20-year-old Greek qualifier Maria Sakkari 7-5, 4-6, 6-3.
It turned out to be an unexpectedly close match between Williams, winner of seven Grand Slam titles and playing in her 19th Wimbledon, and Sakkari, making her first appearance at Wimbledon and third at a major.
Keys, seeded No. 9 after breaking into the top 10 for the first time this month, also reached the third round with a 6-4, 4-6, 6-3 win over Kirsten Flipkens. Keys, a quarterfinalist last year, had 48 winners — more than double the Belgian player's 22.
Stephens, seeded No. 18, defeated China's Peng Shuai 7-6 (5), 6-2 in a first-round match. Stephens reached the quarterfinals in 2013.
Boserup advanced when seventh-seeded Belinda Bencic retired with a wrist injury with the American leading 6-4, 1-0.
The 28th-seeded Querrey advanced to a third-round men's matchup against two-time defending champion Novak Djokovic by beating Brazil's Thomaz Bellucci 6-4, 6-2, 6-2. The match had been suspended by rain Wednesday at 5-2 in the second set.
The 27th-seeded Sock beat Robin Haase 6-1, 6-3, 6-7 (3), 6-4. His third-round opponent will be sixth-seeded Canadian Milos Raonic, who hit 25 aces in a 7-6 (5), 6-4, 6-2 win over Andreas Seppi.
The unseeded Johnson reached the third round for the first time after a 6-1, 7-6 (6), 6-3 win over Jerome Chardy.
In a first-round match extended over three days because of rain, the 18th-seeded Isner downed Marcos Baghdatis 7-6 (2), 7-6 (5), 6-2. Isner, who had 33 aces, has yet to advance past the third round at the All England Club.
"The long days in the locker room the last days were just brutal," Isner said. "You can only play so much putt-putt in the locker room with your friends and what-not."
Young, meanwhile, won his first match at Wimbledon after losing in the first round in his five previous appearances. He beat Argentina's Leonardo Mayer 6-4, 7-5, 3-6, 6-3.
In other men's play, fifth-seeded Kei Nishikori came from behind to beat Julien Benneteau 4-6, 6-4, 6-4, 6-2 on Centre Court; and No. 9 Marin Cilic needed four sets to beat Sergiy Stakhovsky 6-2, 6-7 (6), 6-4, 6-4.
British player Dan Evans beat 30th-seeded Alexandr Dolgopolov of Ukraine 7-6 (6), 6-4, 6-1. Evans will next face seven-time champion Roger Federer.
No. 13 David Ferrer, a two-time quarterfinalist, was eliminated by Nicolas Mahut in straight sets, 6-1, 6-4, 6-3. And No. 23 Ivo Karlovic was upset by Slovakian qualifier Lukas Lacko, 6-3, 3-6, 7-6 (5), 6-4.
In women's matches, fourth-seeded Angelique Kerber advanced with a 6-1, 6-4 win over Varvara Lepchenko, and No. 5 Halep beat Francesca Schiavone 6-1, 6-1. No. 14 Samantha Stosur was knocked out 6-4, 6-2 by 2013 runner-up Sabine Lisicki.