Former world number one Hewitt has not won an ATP Tour trophy since the pre-Wimbledon event in Halle in 2010 when he stunned Roger Federer in the final.
But he could not repeat the feat against top seed Isner who relied on his big serve to carry him to a 7-6 (7/1) 6-4 victory in one hour and 32 minutes.
After both players failed to capitalise on their one break point chance in the opening set, Isner built a 4-0 lead in the tie-break and closed it out on his first of five set points.
He broke Hewitt in the third game of the second set, and after the Australian saved one match point on serve in the ninth game, Isner clinched the victory with his 16th ace.
Despite the loss, it was an encouraging week for Hewitt, who has slipped to 233 in the rankings after battling to overcome a toe injury.