Isner struggled through a number of unforced errors and at least a handful of double faults. Istomin of Uzbekistan, ranked 63rd in the world, broke Isner in the seventh game of the first set as Isner hit two shots into the net and sailed a backhand long in the game.
After gaining another break in the fifth game of the second set, Istomin won the set at love on Isner's serve, game point coming on a cross-court passing shot with Isner at the net.
Earlier in the day, fourth-seeded Sam Querrey of the United States defeated Alejandro Falla of Columbia 6-3, 6-3 to advance to the tournament's second round.
Fifth-seeded Kei Nishikori of Japan played Tuesday's late match against qualifier Illya Marchenko of Ukraine.
In the women's draw, third-seeded Sabine Lisicki of Germany and seventh-seeded Magdalena Rybarikova of Slovakia both won their matches in straight sets. Lisicki defeated qualifier Madison Keys of the United States 7-5, 6-3, while Rybarikova beat American Lauren Davis 7-6 (2), 6-2.