The former world No.1, determined to resurrect his season and build on a promising showing in Monte-Carlo, suffered a second round loss to Martin Klizan.
The Slovakian, on the comeback trail after injury, upset the 12-time major champion 6-2 1-6 6-3 to ensure their will be no last eight meeting between Djokovic and Rafael Nadal.
Nadal, the defending champion, began his title defence with a 6-4 6-4 win over compatriot, Roberto Carballes Baena
Klizan has slipped from view in the last 12 months, but he remains a formidable foe when focused and inspired. If the 28-year-old had the discipline and composure of his contemporises, his trophy haul would be considerably greater than five.
It’s another setback for Djokovic – and he has much to ponder ahead of next month’s Madrid Masters. The 30-year-old has picked up just five wins from his ten matches this year.
It was, once again, a difficult draw for Djokovic, and the severity of his task was made clear in the early exchanges.
Klizan cracked his menacing lefty forehand with unerring accuracy in the opener, however it was not just his brawn that helped him pocket the set by six games to two. His use of the dropshot was also highly effective, and his opponent was forced to dance to his tune.
Djokovic responded impressively and began to dictate matters from the baseline. The winners were less frequent from Klizan, and he was soon embroiled in a decider.
Klizan stayed with his opponent in the early stages of the third set before making his moving in the eighth game. The world No.140 carved open two break-points – and he converted his first opportunity after some indecision from his rival.
Djokovic hesitated when faced with a high forehand volley at the net, and instead of opting for power; he tamely nudged the ball into the hitting zone of his grateful rival.
Klizan threaded a backhand passing-shot down the line to secure a pivotal breakthrough, and he was celebrating a first ever victory over Djokovic moments later.
Elsewhere on the grounds, last year’s finalist, Dominic Thiem, overcame a first set challenge from Jaume Munar before prevailing 7-6(8) 6-1. Grigor Dimitrov is also through after a comprehensive 6-2 6-1 success over Gilles Simon.
Kei Nishikori's stay in Barcelona is already over. The weary looking Japanese, who reached the final at the Monte-Carlo Masters last week, retired while trailing Guillermo Garcia-Lopez 6-3.