The five-time champion, who has now won all bar one of 40 matches at Roland Garros, played a five-set match at the Paris venue for the first time in seven visits.
"Always it's a very, very close match against John because, with his serve, he arrives to the tie-break and you play under pressure all the time," Nadal said. "For moments in the tie-break I didn't play very well. I was probably too nervous."
Nadal last lost a set at the French Open in 2009, when he was eliminated by Robin Soderling in the fourth round. He had never previously dropped a set in the first or second round. The top-seeded Nadal is trying to equal Bjorn Borg's record of six French Open titles.
The Spaniard came into the tournament after losing to Novak Djokovic on clay in finals in Rome and Madrid. But the top-ranked Spaniard is still considered by many to be the favourite at Roland Garros.
That didn't seem to matter - at first anyway - to Isner, the man best known for playing in the longest tennis match in history last year at Wimbledon.
Despite losing the first set, the tall American held strong and forced Nadal into a pair of tiebreakers, where his big serve helped him put Nadal on the defensive. But with only six unforced errors the rest of the way - none in the fourth set - Nadal proved he is still the man to beat on clay.
Fifth-seeded Soderling beat American Ryan Harrison 6-1, 6-7 (5), 6-3, 7-5. Harrison won a spot in the main draw after Benjamin Becker withdrew.
Of the other seeds in action yesterday, No 8 Jurgen Melzer, of Austria, No 20 Florian Mayer, of Germany, No. 21 Alexandr Dolgopolov, of Ukraine, and No 24 Sam Querrey, of the United States, also advanced, but No 11 Nicolas Almagro, of Spain, lost to Polish qualifier Lukasz Kubot.