The third-seeded Isner bested his Czech opponent 3-6, 6-4, 7-6 (11-9), remaining perfect in the event, which is held in his home state of North Carolina.
He improved to 10-0 all-time at Wake Forest University with the victory.
After going down 1-3 in the tiebreaker, Berdych proceeded to win four straight points. But he failed to convert on three championship point attempts, while Isner used a blistering forehand winner to wrap it up on his second championship point try.
The crown was the second this year for Isner, who also won in Newport last month. He fell in the finals at Indian Wells and Houston earlier this season.
The second-seeded Berdych came up a bit short in his bid for an eighth career title and first in the United States.
He fell to 7-8 all-time in ATP finals, including a 1-2 mark this year after winning in Montpellier in February.