The former V8 Supercar champion looked on course to his maiden Sprint Cup win on Sunday at Infineon Raceway but his fuel-saving tactics ended up costing him big in the closing laps of the race.
Ambrose was shutting down the engine of his car to save fuel under the final caution with six laps to go, when he suddenly struggled to get it going again, rolling to a halt on the way up to turn two.
Around four seconds later he was able to fire the engine back on, but already six cars had gone by him before he was able to get back up to caution speed, which was set at 40 mph for this event.
NASCAR ruled that Ambrose had failed to maintain "reasonable speed" during the caution and despite him driving back up to the lead, he eventually had to drop back to seventh for the last restart, following instructions from officials.
"I'm disappointed," said Ambrose following the race. "It's NASCAR's house and I'll always play by the rules. I don't agree with it, I don't like it and that's only because I lost the race...
"I had the motor turned off trying to save a bit of fuel and just had trouble getting it fired again. That's it."
The 33-year-old admitted his mistake in the end, saying he should have had the engine fired up before going up the hill into turn one. Ambrose was trying to save some gas in case the race went beyond the scheduled distance, due to the maximum of three green-white-chequered finishes allowed by the rules for this year,
"I tried to refire it on that left-hand uphill and it just didn't refire," said Ambrose. "My bad. Really disappointed. I don't like the call but I should have had the motor cranked up and it would have never been an issue."
NASCAR's Robin Pemberton said the ruling indicates that once a car gives up position by stopping on the track during a caution, it is not allowed to get any places back behind the pace car.
"For whatever reason, he stopped on the race track," Darby said. "And when you stop on the race track, you relinquish whatever position you're in whether it's 30th or whether it's first. Once you get going again, you blend back into line."
Race-winner Jimmie Johnson, who dominated the first part of the race, was second when Ambrose stopped on track and then led the field to the final restart, pulling away to claim his first Sprint Cup series win on a road course.
However he felt some sympathy for the Australian and his team, who proved they would have been hard to beat had they stayed at the front for the final sprint.
"I feel bad for him and his team owner," said the reigning champion. "His team owners gave me my chance in Nationwide in '98 maybe it was. So I'm very familiar with the team.
"I think Marcos had a very fast car in the short runs. I had a try or two at him before that, couldn't get by him. So I'm not sure I would have gotten by him. It was definitely a gift kind of handed to us, as [crew chief] Chad [Knaus] said on the radio to me. From that point on, I just needed to get a good restart and get away from those guys."
Ambrose dominated every practice session of the weekend but also made a mistake in qualifying, as his foot got stuck between the throttle and brake pedals as he approached the final turn on his flying lap.
Restarting seventh for the last five laps, he eventually finished sixth behind Jeff Gordon.
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