The retirement came three years after Isner beat Nicolas Mahut 70-68 in the fifth set in a match that lasted more than 11 hours over three days. Now, Isner is out after one of the shortest matches in Wimbledon history.
Isner, who was trying to reach the third round at Wimbledon for the first time, said he felt something in his knee when he landed on his left leg after hitting a serve on the third point of the match.
"I always serve and land on my left leg, like I have done 20 million times playing this game, and this is the first time I just felt this sharp pain," he said. "It didn't pop. It just grabbed like really badly, and I knew I was in serious trouble then. I knew at that point it was not likely I was going to be able to play."
Isner said it appeared to be a tendon injury, not a major tear, and he does not expect to need surgery.
"I just can't bend my knee," he said. "I can walk as long as I keep it straight."
Away from the injury front, seven-time champion Roger Federer was to face Sergiy Stakhovsky on Centre Court, while 2012 U.S. Open champion and Wimbledon runner-up Andy Murray was placed on Court 1.
Maria Sharapova, the 2004 Wimbledon champion, was scheduled to play her second-round match on Court 2 against 131st-ranked qualifier Michelle Larcher de Brito of Portugal.