The U.S. Post Office gave Cheyenne's aviation its first boost. With the introduction of airmail routes after World War I, Cheyenne civic leaders lobbied to establish Cheyenne as a stop. Buck Heffron piloted the first air mail flight to Salt Lake City on 8 September 1920. He flew a DH-4 that could barely get high enough to clear the mountains and had a maximum speed of 100 mph (160 km/h). The pilot was one of the brave aviators who was guided by a few instruments, maps and landmarks. By the latter 1920s, Cheyenne had become an important crossroads for mail and passenger flights on a transcontinental route between San Francisco and New York as well as a north–south route from Cheyenne to Pueblo, Colorado. Cheyenne was chosen over Denver due to the mountain peaks exceeding 14,000 feet immediately west of Denver. The initial carrier operating on the transcontinental route was Boeing Air Transport. By the early 1930s, Boeing had merged with three other carriers to form United Airlines.

Cheyenne's airport saw its first paying passengers in the 1920s. The first was Elizabeth Brown, a female barber. She enjoyed a ride with World War I pilot, C.A. McKenzie, in a Curtis Oriole biplane. With the step up to the DC-3 in 1937, passengers enjoyed greater comfort and safety. Soon United DC-3s were flying Cheyenne passengers to both coasts and south to Denver.

The Boeing/United Airlines Terminal Building, Hangar and Fountain, built for what would become United Airlines between 1929 and 1934, are listed on the National Register of Historic Places.

During World War II the airport was a completion and modification center for B-17s. Captain Ralph S. Johnson was a test pilot for the then United States Army Air Corps, forerunner to the United States Air Force. The tail turret on the B-17 is known as the "Cheyenne" turret because it was invented at Cheyenne. United Airlines maintained its Douglas DC-3s at Cheyenne; in 1946 it had 1400 employees based locally. Until 1961 the airport was the training center for United Airlines stewardesses from across the country.

The airport was visited by Charles Lindbergh, aboard the Spirit of St. Louis, and Amelia Earhart. Many historic events are chronicled on the walls of the airport restaurant. One of the airport's celebrated visitors in recent times was child aviator Jessica Dubroff, who lost her life when her small plane crashed after takeoff in terrible weather in April 1996.

Because of its high altitude, aircraft manufacturers test their new jetliners at Cheyenne. Recent tests have involved Embraer of Brazil's E-170 and E-190 aircraft as well as Boeing's 737-900 and 787 Dreamliner jets and most recently, the Boeing 737 MAX.

The airport terminal contains plaques of the inductees into the Wyoming Aviation Hall of Fame. The 2013 inductee, Raymond A. Johnson, lived primarily in Cheyenne after 1960.

In November 2018, Cheyenne Regional Airport's current passenger terminal opened. The former terminal has since been proposed to be repurposed as an aviation museum.

A jet bridge was installed at the airport's terminal in 2023, at a cost of $2 million.