Isner had said he was playing with house money after coming within a whisker of defeat on Wednesday, and No 7 seed Johnson collected today, winning 6-3 5-7 7-6 (7-3).
While 208cm Isner's powerful first serve remained a weapon, it was when he was forced into second serves that Johnson pounced.
The 27-year-old Johnson consistently pushed inside the baseline to return and during rallies to put the pressure on world No 19-ranked Isner under a sunny Auckland sky.
Johnson forced Isner to save nine of 10 break points as he broke in the first set to win it and, after losing the second, threatened to break again early in the third.
But, in the only quarter-final to feature a clash between two seeds, Johnson eventually took his chance to claim the match in the tiebreaker when his forehand nicked the net and pulled up too short for Isner.
The No 2 seed's exit adds to the loss of other big names, who on Wednesday tumbled out of the tournament.
It started with defending champion and top seed Roberto Bautista Agut pulling out with a stomach bug, while sixth seed Feliciano Lopez also withdrew and No 3 seed David Ferrer crashed out.
Johnson meets the winner of the clash between US No 4 seed Jack Sock and Frenchman Jeremy Chardy in the semi-finals.
Cypriot Marcos Baghdatis has beaten former champion Jiri Vesely in straight sets to also reach the last four.
The eighth-seeded Baghdatis mixed his shots well today and was never challenged on serve as he won 6-2 6-4.
He will face an in-form Portuguese world No 44 Joao Sousa in the semi-finals. The 27-year-old Sousa accounted for 58th-ranked Dutchman Robin Haase earlier on Thursday 6-3 6-2.
