Mackintosh, who celebrated his 24th birthday last Friday, has strained the meniscus in the back of his left knee.
He will be forced to rest for six weeks.
Mackintosh suffered the injury in the first half of the first round game at Carisbrook against the Brumbies.
There had been hope he could play on Friday night against the Hurricanes, but he will not now be sighted until April at the earliest.
It is a huge blow for the Highlanders, already plagued by players not being available, just two weeks into the Super 14 competition.
Four key players did not take the field in the narrow loss to the Hurricanes in Wellington on Friday night.
Mackintosh and Jimmy Cowan (elbow) were both injured against the Brumbies.
Lock Tom Donnelly injured his thumb in pre-season training and has yet to play in the Super 14 this season.
Winger Fetu'u Vainikolo was stood down from Friday night's game for disciplinary reasons.
Mackintosh had made the All Blacks last season and was expected to be a big contributor for the Highlanders this year.
He will likely not be back until Easter, when the Highlanders take on the Reds in Invercargill.
Highlanders coach Glenn Moore said the loss of Mackintosh was obviously a big blow but the side just had to get on with the rest of the season.
Cowan, who is also the side's captain, had made progress with his sprained right elbow, but it was still unclear whether he would be able to play against the Waratahs in Sydney on Friday night.
A decision on whether he plays is likely to be made before the team leaves on Wednesday.
Inside back Mathew Berquist rejoined the team on Saturday, after taking leave to be with his sick mother.
Donnelly may be available to play against the Crusaders on March 7.
Moore said there was the usual bumps and bruises from Friday night and said spirits had picked up after the heart-breaking loss to the Hurricanes.
The Highlanders conceded two tries in the final five minutes to go down 22-17.
Moore said after reviewing the tape of the game, the side blew it near the end, having the strategies in place in the final minutes but failing to close the game down.
The Highlanders will face an unbeaten Waratahs side after the Australian side beat the Chiefs 11-7 in a scrappy match in Sydney on Friday night.
