The five-time champion, who improved to 39-1 at Roland Garros, played a five-set match at Roland Garros for the first time in seven visits.
"Tough, tough moments for me," Nadal said. "I played too nervous, in my opinion."
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.
Nadal 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 favorite 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.
"I didn't play well in the tiebreak, and I didn't have chances to have the break," Nadal said. "When you play against these kind of players, the pressure is there all the time. You have to play all the time very safe."
Sharapova, who was coming off the biggest clay-court title of her career, rolled into the second round by beating Mirjana Lucic of Croatia 6-3, 6-0.
The seventh-seeded Russian won the Italian Open before coming to Roland Garros to seek the only Grand Slam title she has never won.
"If you feel pressure, it's part of the business. It's part of the sport," Sharapova said of the expectations placed on her. "That means there's something on the line. That means you want it bad. It's how you handle it.
"Without pressure, what's the sport about? It's how you really handle the situation."
Against Lucic on center court, Sharapova was not troubled. She won nine straight games to finish the match and never faced a break point.
The sixth-seeded Li, who became the first Chinese player to reach a Grand Slam final, dominated at times and struggled at times in her 6-3, 6-7 (6), 6-3 win over Barbora Zahlavova Strycova of the Czech Republic.
But first on Court Philippe Chatrier, an emotional Virginie Razzano played despite the death of her fiance eight days ago. The Frenchwoman, playing with a black ribbon on her shirt, lost to 24th-seeded Jarmila Gajdosova of Australia 6-3, 6-1.
"I felt a lot of emotion, a lot of pain on court today," Razzano said. "The pain is permanent within me. It's very hard. But it felt good to be surrounded by so many people and to be here.
"I tried to pay tribute to Stephane today. It was almost a 'mission impossible,' but I did my best."
Razzano's fiance and coach Stephane Vidal died on May 16 after being diagnosed with a brain tumor nine years ago. He was 32.
The 28-year-old Razzano said last week that she asked Vidal before he died if she should play at Roland Garros and he said, "Yes, you need to play."
Razzano will also play in the doubles tournament with partner Alize Cornet.
Murray made it through to the second round by beating French qualifier Eric Prodon 6-4, 6-1, 6-3.
"He didn't want to have any long rallies, so he was hitting a lot of drop shots, going for shots. He'd change the rhythm or change the pace of the ball a lot," Murray said. "I was annoyed with the way I was moving. I was hitting the ball from the back of the court, especially towards the end of the match, and served well, but didn't move particularly well."
The fourth-seeded Briton went on a four-match winless run after losing the Australian Open final, but he reached the semifinals at clay-court warmups in Monte Carlo and Rome.
The fifth-seeded Soderling beat Ryan Harrison of the United States 6-1, 6-7 (5), 6-3, 7-5. Harrison won a spot in the main draw after Benjamin Becker withdrew.
No. 8 Jurgen Melzer of Austria, No. 16 Fernando Verdasco of Spain, No. 20 Florian Mayer of Germany, No. 21 Alexandr Dolgopolov of Ukraine, No. 24 Sam Querrey of the United States and No. 32 Kevin Anderson of South Africa also advanced, but No. 11 Nicolas Almagro of Spain lost.
Based on reporting by the Associated Press.