The Sharks (46-27-9), who won their final two games after going 1-8-1 in their previous 10 games, finished in second place in the Pacific Division and will have home ice against the Vegas Golden Knights in the first round.
Evander Kane, Gustav Nyquist and Brent Burns each had a goal and an assist for San Jose.
"Any time you get (101) points, it's a good year. It's a lot of work we put in," said Sharks coach Peter DeBoer, who won his 400th NHL game. "It's been a tough last month with some of the injuries and adversity that we've had, but I think when I look at the whole season, we put in a lot of work and it's a big accomplishment. All that work really is just to get a ticket to play next week."
Jost gave Colorado a 1-0 lead at 2:56 of the first period, getting his own rebound in the high slot after his initial shot was blocked by Barclay Goodrow.
Burns tied it 1-1 at 7:25 on a shot from the right point after Tomas Hertl won a face-off in the right circle, and Kane put San Jose in front 2-1 at 11:15, jamming in the rebound of Nyquist's shot from the left circle.
MacKinnon tied it 2-2 at 1:29 of the second period. After taking a return pass from Gabriel Landeskog, he split San Jose's defense and put a forehand shot inside the right post.
Kevin Labanc gave the Sharks a 3-2 lead at 6:57 after his shot deflected up off the stick of Colorado defenseman Patrik Nemeth and over Varlamov.
Nyquist extended the lead to 4-2 at 5:38 of the third period, scoring short side from the bottom of the right circle following a turnover by Nemeth.
Joe Pavelski appeared to score into an empty net at 16:59, but Avalanche coach Jared Bednar challenged for goaltender interference, and the call was reversed after a video review.
However, Micheal Haley scored an empty-net goal with 45 seconds remaining to make it 5-2.
Sharks defenseman Erik Karlsson had two shots and was plus-3 in 22:01 of ice time in his return after missing 17 games because of a groin injury.
"I thought he looked good for not playing in a long time," DeBoer said. "He handled pucks, he didn't look real rusty with the puck. His legs felt good, so I thought it was a good step. It was nice to get him in there. Even a rusty Erik Karlsson is still a pretty good player."