Isner, the world number 12 and top seed, defeated fellow American Wayne Odesnik 6-3 7-6 (8-6) to begin his campaign at the Newport grass-court event in style.
After losing in the third round at Wimbledon to Feliciano Lopez, Isner bounced back with 19 aces as he dismissed left-hander Odesnik in one hour and 19 minutes.
Other seeds to advance at the ATP 250 event were Lleyton Hewitt (third), Nicolas Mahut (fourth), Jack Sock (fifth) and Adrian Mannarino (eighth).
Australian veteran Hewitt lost serve five times but battled his way past American Ryan Harrison 1-6 7-5 6-4.
Frenchman Mahut was too good for Argentina's Facundo Arguello 7-6 (7-1) 6-4, while American Sock - fresh from winning the Wimbledon men's doubles title with Vasek Pospisil - posted a 6-4 6-3 win over compatriot Alex Kuznetsov.
Mannarino, of France, downed Chinese Taipei's Jimmy Wang 6-2 6-4 to reach the last 16.
Ukraine's Sergiy Stakhovsky surpassed Uzbekistan's Farrukh Dustov 6-3 6-2, while qualifier Austin Krajicek won his first tour-level match when he defeated American compatriot Tim Smyczek 7-6 (9-7) 6-1.
Croatian qualifier Ante Pavic beat Romanian Marius Copil 7-6 (7-0) 6-4, and Australian qualifier Luke Saville got past Canada's Peter Polansky 6-4 6-2.