The pair were fastest all week and overcame a mid-race tyre failure to claim the win.
Cam Waters and James Moffat finished second, with Brodie Kostecki and David Russell rounding out the podium.
"I don't know what to say, this is unbelievable," said Holdsworth.
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It's Holdsworth's first win at the Bathurst 1000, while Mostert added to his 2014 title.
"It was a pretty tough race, when we did that tyre I thought it was going to be a pretty tough slog," Mostert said.
"We started pole and we won the race, every time you start on pole you never think you're going to win."
Tony D'Alberto got the jump off the start line to lead Holdsworth and Garth Tander into the first corner, but even before the field had completed a single lap he'd been shuffled back to third.
Holdsworth took the lead into The Chase for the first time, while Tander slipped inside the DJR Mustang at Murray's Corner.
Moffat runs across the grass at The Chase
Across the line for the first time Holdsworth had the lead from Tander and D'Alberto, while the Team 18 Commodore of James Golding suffered the race's first drama.
Golding ended up on the grass at The Chase on lap two, and pitted immediately afterwards, reporting a power steering problem with the car.
Holdsworth had extended his lead to nearly five seconds by the ninth lap, while Tander in second was seven seconds clear of D'Alberto and Tim Slade, as the leading two cars opened up an early gap.
Thomas Randle brought out the first safety car of the race on lap 17 when he found the sand trap at Turn One, wiping out the seven second lead Holdsworth had built.
Power steering failure claims Perkins
The leaders took the chance to pit, with Holdsworth remaining behind the wheel of the 25 car, while Waters replaced Moffat, who had endured a difficult first stint, dropping from third to 12th.
The second DJR car of Will and Alex Davison also suffered, having to double stack behind the team's other car, dropping the Davison brothers from fifth down to 18th.
Lap 30 saw the Will Brown/Jack Perkins car suffer a major power-steering failure, as the car starting smoking heavily on the run across the top of the mountain while running in ninth position.
Perkins was forced to pit for repairs, which cost the duo 12 laps, effectively ending their day, although they continued to circulate once it was fixed.
Tyre failure for Chaz Mostert
All the while the lead Walkinshaw Andretti United car of Lee Holdsworth carried on in the lead, eventually pitting on lap 38 to change to Chaz Mostert.
On lap 49, however, Mostert suddenly slowed as he made the run onto Conrod Straight, with a left-rear tyre failure. His 12 second lead evaporated as he headed for the pits, fortunately without any serious damage apart from loss of time. He rejoined in 12th spot, eventually working his way back into the lead as those in front pitted, but he was out of sequence with the rest of the field.
That was how the next 40 laps played out, with Mostert and Holdsworth displaying serious pace in the WAU Commodore, but pitting at different times.
It resulted in Holdsworth holding a 38 second lead when he pitted on lap 92, with Moffat in second with Tander in close company in third and D'Alberto rounding out the top four.
Safety car caused by echidna
With 56 laps to go came on of the most unusual safety car interventions in Bathurst 1000 history, when an echidna wandered onto the track.
"This place is just mad, isn't it," said Mark Skaife.
As the saying goes, safety cars breed safety cars, and so it was again this time, when Jayden Ojeda crashed just after the race went green again.
That caused a rush for the pit-lane, with the Triple Eight cars of Shane van Gisbergen and Jamie Whincup running first and second. The stop was disastrous for Whincup, who was forced to double stack, with the team only able to service a single car at a time, dropping him out of the top 10.
Will Davidson and Whincup made contact as they both left the lane, with officials assessing Davidson a five-second penalty.
Feeney finds the wall at Bathurst
The race had only just restarted when Jake Kostecki found the wall at Forrest's Elbow, necessitating another safety car, but importantly removing any need to fuel save to get to the finish.
Mostert was able to pull away from van Gisbergen when racing resumed, quickly opening up a three-second lead.
Both Mostert and van Gisbergen had completed their final stops when Anton de Pasquale ground to a halt coming out of The Cutting, which brought out the safety car yet again. The DJR driver complained that he could change gear, but had no drive, and his day was done.
The rest of the field headed for the pits for the final time, leaving a 20-lap shootout to the finish, with Mostert leading van Gisbergen and Waters.
But the safety car wasn't done yet, with Broc Feeney finding trouble at the top of the mountain, crashing out of ninth place.
When the race went green again, Mostert was once again able to open up a four second lead, but there was one last twist, when van Gisbergen's right front tyre delaminated, forcing him to pit with just a handful of laps remaining.
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