The top-ranked Serb wasted a chance to serve out the match and was beaten 2-6, 7-5, 6-4 by sixth-seeded Tomas Berdych in the Italian Open quarterfinals on Friday.
It's the second consecutive week that Djokovic has struggled, having also lost to Grigor Dimitrov in the second round of the Madrid Open, where he was bothered by a right ankle injury.
Djokovic said he had been playing pain-free this week.
Still, he will need to get his game in shape in a hurry with the French Open, the year's second Grand Slam, starting on May 26.
"I know I can play well on clay and so I shouldn't allow myself to have these drops and let's hope it won't happen for Roland Garros,'' Djokovic said of the Paris tournament, the only major he has not won. "I'm fine. I just lost my rhythm.''
Djokovic served for the match at 5-3 in the second set but Berdych ripped a backhand winner that landed on the line on his first break point and won the final four games of the set.
"It's a very strange situation,'' Djokovic said. "I played very well and was two points from winning. Then the entire match changed and I was a different player.''
In the semifinals, Berdych will face either six-time Rome champion Rafael Nadal or fourth-seeded David Ferrer, who were up next on center court.
In the women's draw, top-ranked Serena Williams rolled past Spain's Carla Suarez Navarro 6-2, 6-0 to extend her winning streak to a career-best 22 matches.
Williams had twice won 21 in a row before, although both of those streaks came a decade ago, in 2002 and 2003.
"I've been pretty consistent, and pretty healthy - knock on wood,'' Williams said. "I feel great - never felt better.''
Martina Navratilova established the longest women's win streak in the Open Era at 74 matches in 1984.
In the other half of the women's draw, two-time defending champion Maria Sharapova withdrew before her quarterfinal against seventh-seeded Sara Errani due to illness.
Williams will next face either two-time Rome winner Jelena Jankovic or Romanian qualifier Simona Halep.
Williams' only title here came in 2002, when she beat Justine Henin in the final. That was also the year she won her only French Open title. Last year in Paris, Williams suffered her first ever opening-round loss in a major, falling to 111th-ranked Virginie Razzano of France.
Sharapova said he was struggling with an illness she first contracted last week, when she was runner-up to Williams in Madrid, although she showed no problems Thursday as she beat 16th-seeded Sloane Stephens 6-2, 6-1.
"I was already a bit off in Madrid and played though it and it came back last night and I got sick again and it's not smart to compete again,'' said Sharapova, adding that she had a fever after beating Stephens. "Obviously with the French Open coming up it is important to make smart decisions.''
The withdrawal meant that Errani advanced to the semifinals. The Italian's opponent will be either third-seeded Victoria Azarenka or ninth-seeded Sam Stosur.
The last Italian to reach the semifinals in this tournament was Raffaella Reggi, who won the 1985 event in the southern city of Taranto.
In other men's play, 36th-ranked Benoit Paire of France routed Marcel Granollers of Spain 6-1, 6-0 to reach his first Masters series semifinal. He'll next face either 17-time Grand Slam winner Roger Federer or rising Polish player Jerzy Janowicz, who were playing a night match.
It's the first time Berdych has reached the semifinals in eight appearances in Rome, having been eliminated four times in the quarters.
The tall Czech took control of the match with his big first serve, and by coming forward to angle shots into the corners, which set up easy volleys.
Djokovic had trouble returning Berdych's first serve and also complained about the court's surface early on.
In the third set, Berdych went ahead with an early break and then shook off some nerves to close the match out on his fourth match point with a big serve out wide that Djokovic could barely get his racket on.
As overcast skies gave way to sunshine, the court dried out, favoring Berdych's power game.
"I lost the concentration,'' Djokovic said. "I started playing more defensively. ... He started playing more confidently, stepping into the court and taking advantage of his opportunities.''
It was only Berdych's second win in 15 career meetings with Djokovic, with his only previous victory coming in the 2010 Wimbledon semifinals.
It was also only Berdych's fourth win vs. a top-ranked player in 20 attempts, with all three previous wins coming over Federer.
"He has been for quite a few years just behind the top four,'' Djokovic said. "He's able to play big matches. He has proven that. So (he) is definitely a very dangerous player on any surface with a serve like that.''
Djokovic was 148-4 when winning the first set since 2011, according to the ATP.