Germany's Brown posted a 6-4 6-7 (7-9) 7-6 (7-4) victory over the ninth-ranked American at the ATP 250 event in Houston.
Brown broke serve in the fifth game of the first set, but that would be the only joy for the receivers all match.
The break was enough to seal the first set for Brown, before he saved two set points in the 10th game of the second set as he forced a tie-break.
Isner, though, showed resolve to save two match points in the tie-break to force a decider.
Brown was showing signs of cracking, offering break-point chances in the first and third games of the deciding set only to hold his nerve and stay in front.
A six-deuce game at 3-3 saw Brown under more pressure, but he did not give Isner a look at break point, before both held serve with relative ease thereafter to send the match to a deciding tie-break.
Brown led 5-1 at the change of ends, but lost three consecutive points to see it back 'on serve'.
Isner, though, could not get his nose in front, losing both points on serve to see his title defence ended at the first hurdle after he received a first-round bye.
Brown's win saw him into a quarter-final meeting with another American, Sam Querrey, who dismissed Australian seventh seed Lleyton Hewitt 6-3 6-4.
In first-round affairs, sixth seed Juan Monaco edged out India's Somdev Devvarman 7-6 (7-4) 6-2.
Hewitt's compatriot Matthew Ebden did not fare any better, as Colombia's Santiago Giraldo handed him a 6-1 6-2 thrashing.