The Argentine stretched his winning streak at the event to 14 straight matches with a 3-6, 6-1, 6-2 decision over the eighth-seeded Isner at FitzGerald Tennis Center.
Del Potro also won the Citi Open in 2008 and 2009.
Isner, meanwhile, ran out of momentum after posting a triumph last week at the BB&T Atlanta Open as his eight-match winning steak was snapped.
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