Although accurate and powerful, with a high muzzle velocity, it was very heavy, with a low rate of fire, which limited its production.

Powerful and accurate for its day, it was carried primarily on the Henschel Hs 129 ground-attack aircraft, commencing in late 1941.

Fed by a 10-round (early versions) or a 30-round box magazine, the MK 101 could penetrate 75 mm (3 in) of armor at 300 m (330 yd) range.

The dozen examples created of the Heinkel He 177A-1/U2 Zerstörer ("Destroyer") experimental prototype attack aircraft used twinned MK 101s on a forward-aimed limited-traverse mount, located in the extreme lower nose at the front of a dramatically enlarged Bola inverted-casemate gondola emplacement under the nose, and was intended for anti-ship and possible "train-busting" use, but was never deployed in combat.

When it was used from beneath an Hs 129's central fuselage as a ventral gun pod-mounted anti-tank weapon, the MK 101's tungsten-cored AP round was capable of penetrating the turret and side armor of the Soviet KV-1 heavy tank.