The first of two C-141A prototypes flew in December 1963. The type entered service in October 1964 and reached initial operational capability in April 1965, soon providing impressive confirmation of its capabilities on the air bridge service to South-East Asia.

During the 1970s, 270 of the surviving 274 C-141s were cycled through an upgrade programme that added a fuselage stretch of 7.11 m (23 ft 4 in) and in-flight refueling capability for true global airlift capacity. The overall cargo capacity of the resulting C-141B was increased by over 30 per cent, and the programme thus added the equivalent of 90 new aircraft in terms of capacity at low relative cost. The YC-141B prototype conversion made its first flight on 24 March 1977, and Lockheed completed the final C-141B on 29 June 1982.

Throughout its career the StarLifter has been a workhorse of the USAF, flying regular supply missions around the world in addition to undertaking special requirements. Of inestimable value to the USAF is the StarLifter's sheer versatility; it can be rapidly reconfigured for many missions. Thirteen C-141B transports of the 437th Air Wing are equipped for the Special Operations Low Level (SOLL) support role, with increased survivability measures, including a forward-looking infra-red turret beneath the nose, improved electronic countermeasures systems and self-defense systems.

Between 1997 and late 1999, the USAF upgraded 64 C-141s to C-141C standard exclusively for Air force reserve command. These received glass cockpits, GPS, an all-weather flight control system and a defensive system incorporating missile warning receivers. The intensive utilization of the C-141 fleet has extracted a heavy toll; the type is being rapidly replaced by the Boeing C-17A. It was withdrawn from front-line service.