The first prototype flew on 27 December 1936. Production Pe-8 were well armed, carrying two 7.62mm machine-guns in a spherical nose turret, two machine-guns or a 20mm cannon in a dorsal turret, one hand-operated 12.7mm machine-gun in the rear of each inboard engine nacelle under the wing trailing edge and a 20mm cannon in a tail turret, plus up to 4,000kg of bombs stowed internally.

Initially the Pe-8 was powered by four 820kW M-105 engines and entered service in this form in 1940. The 1941 model had 1,080.5kW Mikulin AM-35A fitted. During the production run (which lasted until 1944) several other engines were installed either experimentally or as a standard type, including the 967kW AM-38, 1,304kW M-82 and a diesel engine.

On the night of 21 July 1941 German bombers attacked Moscow for the first time. As a reprisal Soviet IL-4 bombers made their first attack on Berlin on the night of 7 August. The resulting damage was minimal but it cleared the way for further attacks. In the spring of 1942 the Soviet ADD or Long-Range Aviation was formed, using the Pe-8 as its main component. Pe-8 raids on Germany began in July 1942 with small numbers of aircraft attacking Konigsberg and then Berlin and elsewhere. However these were not very successful and extremely heavy losses were endured. Although Pe-8 remained operational as bombers until the end of the war, a number were converted into transports.