Nishikori came into the clay-court grand slam enjoying his highest seeding at a major event, but lost to the Slovakian 7-6 (7-4) 6-1 6-2.
The Japanese had made a great start to the clay season, winning the Barcelona Open in April before reaching the final of the Madrid Open earlier this month.
However, Nishikori retired in the final set of that final with Rafael Nadal because of a back injury, and the 24-year-old appeared short of fitness on his return to action.
It took Klizan just an hour and 57 minutes to complete a victory over a player ranked 49 places above him.
Nishikori had looked set to claim the first set after coming back from 2-0 down to lead 5-3, but Klizan fought to take the opener on a tie-break.
Matters only got worse for Nishikori from that point, the world number 10 landing under half of his first serves as Klizan sealed the next two sets in utterly convincing fashion and set up a second round meeting with Robin Haase, who beat Nikolay Davydenko in straight sets.
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