In 1924, Roy B. "Cap" Dodson started the first airport in the area, located on the north edge of Kirksville. However, an airfield at the present location of Kirksville Regional Airport wasn't created until 1930 when the federal government built "Intermediate Field, Site 12" on the Kansas City-Chicago airway as one of a series of emergency landing strips across the nation.

With America's entry into World War II, the Kirksville Municipal Airport (as it had been declared in the late 1930s) received a major upgrade from the Civilian Pilot Training Program, later renamed the War Training Service. In 1942 a paved all-weather landing strip, hangars, a tower, and a small restaurant were built. Between January 1941 and January 1944, between 1,800 and 2,000 men were trained in the program at Kirksville.

On October 1, 1960, Ozark Air Lines began scheduled flights to Kirksville. The initial Ozark route flew between Kansas City and Chicago with stops in Kirksville, Ottumwa and Cedar Rapids, Iowa, and Moline, Ill. Later in the 1960s, Ozark switched Kirksville service from a Kansas City-Chicago route to a Des Moines-to-St. Louis one.

Airport improvements came after a bond issue was approved by Kirksville voters in November 1967. A new six-thousand-foot concrete runway was built, as well as a new terminal building and improved hangar facilities. With the airport improvements came a new name, Clarence Cannon Memorial Airport—chosen to honor long-time U.S. Congressman Clarence Cannon of Missouri who had done much to help secure air service and funding for the airport. That name was retained until the Kirksville Regional name was adopted in 1984, and the terminal building was named after Rep. Cannon.

Ozark flew its final route to Kirksville on April 23, 1976. Fortunately a local pilot and dentist, Dr. Stephen Barber, had established a small commuter air service, Horizon Airways, in 1972. Horizon was able to help fill the void left by Ozark's departure, eventually expanding by 1976 to offer service to both Kansas City and St. Louis, and changing its name to Air Missouri.

From 1978 to 1987, Kirksville was served by Green Hills Aviation with commuter flights to St. Louis. From 1987 through December 1989, Midcontinent Airlines flew to Kansas City as Braniff Express on behalf of Braniff (1983-1990). Kirksville then went without commercial service during 1990 until Redwing Airways began commuter flights to Kansas City, which continued until October 2001.

From 2001 through 2006 Corporate Airlines, which later became RegionsAir, provided flights to St. Louis. The carrier first operated as Trans World Express on behalf of TWA, then as American Connection on behalf of American Airlines.

Air Midwest (operating as US Airways Express), a subsidiary of Mesa Air Group, provided flights to Kansas City from November 2006 through May 2008. Kirksville went without service until July 2008, when Air Choice One began scheduled flight operations twice daily, to and from St. Louis.

In September 2010, Cape Air replaced Air Choice One with service to St. Louis that continued until 2023, providing three daily round trips. In 2023 Cape Air was replaced by Contour, which initially provided 12 flights per week to Chicago using regional jet aircraft. This was the first scheduled jet service to Kirksville.