The Buffaloes (5-6) began the season with five straight wins and a No. 19 ranking before dropping six in a row -- including Saturday's 30-7 setback to Utah.
MacIntyre will not coach Colorado's final game this weekend against California. An interim coach will be named, athletic director Rick George said.
"I want to thank Mike for six seasons of hard work and dedication to the program, both on and off the field," George said in a statement. "There's no doubt that the 2016 season was magical, and it appeared we were headed back to taking our place among the nation's elite. However, analyzing the direction of the program over the last two years, I felt this is the necessary time to make a change."
Denver7, an ABC affiliate, reported last week that Colorado would part ways with MacIntyre before George released the following statement:
"We do not comment on speculation or unsubstantiated rumors with anonymous sources. Let me just say I have made no decisions regarding the future of the football program," George said in his statement. "As I've stated in the past, we continually evaluate all aspects of all of our 17 intercollegiate sport programs."
MacIntyre was hired Dec. 10, 2012, and has three years and roughly $9,975,000 remaining on his contract after this season.
When hired, MacIntyre took over a program that was among the worst in the country, having gone 4-21 in two seasons under Jon Embree.
MacIntyre led the Buffaloes to the Pac-12 championship game in 2016 and earned national coach of the year honors. Overall, he owns a 30-44 record.
ESPN reported possible candidates to replace MacIntyre include former Oregon coach and Chicago Bears offensive coordinator Mark Helfrich, Fresno State coach Jeff Tedford, Boise State coach Bryan Harsin and West Virginia coach Dana Holgorsen.