Since the Pe-2's mass production had begun, its defensive armament was recognized by the military as weak for a modern bomber. Beginning with series 13 (launched in April to May of 1941), the ShKAS hatch mount was replaced with a VUB-2 turret with a 12.7mm BT machine gun and a magazine capacity of 200 rounds. The starboard ShKAS machine gun mounted to port forward was replaced with a 12.7mm BK machine gun with 150 rounds. Also, the magazine capacity of the port ShKAS was decreased to 450 rounds.
By June 22, 1941, when Germany invaded the USSR, factories had managed to produce 490 new dive bombers. Subsequent upgrades of the Pe-2 were then made based on combat experience.
Series 22 saw the installation of M-105RA engines with floatless carburettors, which improved the aircraft's behavior when diving. Beginning with series 23, an extra ShKAS machine gun with 225 rounds was installed in the radio operator/gunner's cockpit. It was designed to fire from side ball mounts and through the upper fuselage hatch. The area of the navigator's and the radio operator/gunner's protective armor was increased, and the robustness of the fuel system was improved. The aircraft was equipped with an inert gas filling system, and duralumin tanks were replaced with fiber ones, which were more effective at dealing with bullets. By series 32, the aircraft could be fitted with guide rails to hold 10 RS-132 unguided rockets under the wings. Some Pe-2s were equipped with RO-82 launchers to fire RS-82 unguided rockets backwards, to defend against any fighters attacking from the aircraft's lower rear. Since September 1941, the Pe-2's bomb load was upgraded by adding KMB-Pe-2 bomblet clusters, which enabled the efficient bombing of soft area-type targets.
A total of 1,671 dive bombers were manufactured by four mass production factories during 1941. The new plane served on all front lines from the Barents Sea to the Black Sea. The Pe-2s were received not only by regiments of the Air Force of the Workers’ and Peasants’ Red Army but also by aviation units of the Northern, Baltic, and Black Sea Fleets.
From February 1942 (series 64) on, the side ball mounts were replaced with pivot mounts and the turret's sighting angle was increased. Series 80 saw yet another defensive upgrade: the navigator's and the radio operator/gunner's positions were given upgraded armor.
On 12 January 1942, chief designer V. M. Petlyakov died in an air crash while flying in a Pe-2. His position was filled by his deputy, A. M. Izakson, who was later replaced by another of Petlyakov's colleagues, A. I. Putilov.