Chiefs quarterback Patrick Mahomes, who was named Super Bowl MVP, threw for 286 yards and two touchdowns as Kansas City rallied from a 10-point deficit at the start of the fourth quarter.
The win is the Chiefs' first Super Bowl championship since Super Bowl IV in 1970.
"I didn't play to my liking in the third quarter. But the guys believed in me, gave me confidence and we kept fighting and we found a way to win it in the end," Mahomes said after the game.
"It's amazing just to be here. It's surreal. We'll enjoy this for a long time."
Meanwhile San Francisco coach Kyle Shanahan remained upbeat despite the loss.
"Win or lose, today doesn't change how I feel about our team," Shanahan said. "I'm proud of the guys, what they did all year. I'm proud of what they did today."
After San Francisco opened the scoring with a field goal, Mahomes put the Chiefs in front in the seventh minute with a one-yard rushing touchdown.
San Francisco responded strongly in the second quarter as quarterback Jimmy Garoppolo connected with fullback Kyle Juszczyk on a 15 yard pass to see the scores levelled at 10-all headed into half time.
The 49ers had all the momentum through the third quarter and appeared to have one hand on the Frank Lombardi trophy when Raheem Mostert closed out the quarter with a one yard rushing touchdown.
However, with his side trailing 20-10, Mahomes saved his best for last, connecting on two passing touchdowns to give the Chiefs the lead with 2.44 left to play.
A 38-yard rushing touchdown from Damien Williams a minute later was enough to seal the comeback victory for Kansas City.