The prototype and development aircraft then began an intensive operational testing program including the first delivery to MATS (63-8078) on 19 October 1964 to the 1707th Air Transport Wing, Heavy (Training), Tinker Air Force Base, Oklahoma. Testing continued and a Federal Aviation Authority type certificate was awarded on 29 January 1965. The first delivery to an operational unit (63-8088) was made on 23 April 1965 to the 44th Air Transport Squadron, 1501st Air Transport Wing, Travis Air Force Base, California. Although operational testing continued, due to the United States' military involvement in South Vietnam, the C-141 was soon employed in operational sorties to the combat zone.
On 8 January 1966, following the disestablishment of MATS, all C-141s were transferred to the newly established Military Airlift Command (MAC).
In October 1973, C-141s and C-5s airlifted supplies from the United States to Israel during the 1973 Yom Kippur War as part of Operation Nickel Grass. Over the course of the operation, C-141s flew 422 missions and carried a total of 10,754 tons of cargo.
The first strategic airlift flight of Operation Desert Shield was flown by a MAC C-141 of the 437th Military Airlift Wing out of Charleston AFB, SC, on 7 August 1990. The C-141 proved to be a workhorse airlifter of Operations Desert Shield and Desert Storm, flying 159,462 short tons (144,661,000 kg) of cargo and 93,126 passengers during 8,536 airlift missions.
On 1 June 1992, following the disestablishment of Military Airlift Command, all C-141s and the airlift wings to which they were assigned were transferred to the newly established Air Mobility Command (AMC). Air Force Reserve Command (AFRC) and Air National Guard (ANG) C-141s and units were also transferred to AMC.
On 16 September 2004, the C-141 left service with all active duty USAF units, being confined to Air Force Reserve and Air National Guard units for the remainder of its operational service life. As of 25 September 2005, there were only eight C-141 aircraft still flying, all from the Air Force Reserve's 445th Airlift Wing (445 AW) at Wright-Patterson AFB. In 2004, 2005, and 2006, the C-141s assigned to the 445 AW participated in missions to Iraq and Afghanistan, mostly for the medical evacuation of wounded service members. The last eight C-141s were officially retired in 2006.
In 1994 one of the aircraft at Wright-Patterson AFB was identified by its crew chief as the Hanoi Taxi (AF Serial Number 66-0177), the first aircraft to land in North Vietnam in 1973 for Operation Homecoming in the final days of the Vietnam War, to repatriate American POWs from North Vietnam.
In 2005, Hanoi Taxi and other aircraft were marshalled by the Air Force to provide evacuation for those seeking refuge from Hurricane Katrina. This aircraft and others evacuated thousands of people, including the medical evacuation (MEDEVAC) of hundreds of ill and injured.
With the 5 May 2005 announcement of the retirement of these last eight C-141s, the Hanoi Taxi embarked on a series of flights, giving veterans, some of whom flew out of POW captivity in Vietnam in this aircraft, the opportunity to experience one more flight before retirement. On 6 May 2006, the Hanoi Taxi landed for the last time and was received in a formal retirement ceremony at the National Museum of the United States Air Force, located at Wright-Patterson Air Force Base near Dayton.
15 aircraft including the "Hanoi Taxi" are now on static display at various air museums around the United States, all the remaining airframes retired to the "boneyard" at Davis-Monthan AFB, Arizona have been scrapped.