Mackintosh has been diagnosed with partially ruptured ligaments between his big toe and his foot this week, commonly known as turf toe, and is expected to be out for six weeks.
Mackintosh (25) said he was gutted about the injury to his right foot, and knew at the time it was serious.
The injury occurred in a scrum about 10 minutes into the second half of the match against the Lions last Friday night and Mackintosh immediately left the field.
He had the injury to his other foot in 2008, and missed more than two months.
He joins lock Tom Donnelly - who had surgery on an injured ankle this week - on the sidelines, leaving big holes in the Highlanders engine room.
It is touch and go whether either player will get back on the field for the Highlanders this season, but Mackintosh is aiming to play again.
"The best-case scenario is that I get back and play against the Waratahs in Invercargill. That is about five weeks away so I'll be doing my best," Mackintosh said.
"I'm not much of a watcher. I never have been and I'll find it particularly hard on Saturday night. I was really looking forward to playing in Queenstown and Invercargill."
His foot is not in plaster and rest is the main remedy for the injury.
The Highlanders play the Waratahs on April 30 in Invercargill, so it is a tight timeframe for Mackintosh, the Southland captain, to make a full recovery.
"It is Murphy's law, really. I had just been saying to someone that this is the best my feet have felt over the past four months and then this comes along."
Otago prop Keith Cameron has been called into the wider training squad as training cover with no replacement into the playing squad.
Meanwhile, wider training squad outside back Ryan Shortland had a knee operation on Thursday and is out for six to eight weeks.
He has been replaced in the wider training squad by Andrew Parata, another Otago back.
