During Operation Barbarossa, it was subordinated to the Schutzstaffel (SS) and deployed in German-occupied territories, specifically the Army Group South Rear Area.

Alongside the Einsatzgruppen detachments and the 1st SS Infantry Brigade, it perpetrated mass murder in the Holocaust and was responsible for large-scale crimes against humanity targeting the civilian population.

The goals of the police battalions were to secure the German army's rear-area by eliminating the remnants of enemy forces, guarding prisoners of war, and protecting lines of communications and captured industrial facilities.

[5] When it crossed the German-Soviet border the regiment came under the control of Friedrich Jeckeln, the Higher SS and Police Leader (HSS-PF) for Army Group South in Ukraine.

[7] During the summer months, the battalion took part in joint actions with the 1st SS Infantry Brigade of the Waffen-SS, both providing support and undertaking independent killings.

[4] In September, Police Battalion 45 participated in the murder of Jews in Berdichev, cordoning off the execution site and leading the victims to pits where they were shot by Jeckeln's staff company.

[11] During the massacre at Babi Yar on 29–30 September 1941, the personnel of Police Battalion 45 cordoned off the area, while Sondercommando 4a and a platoon of Waffen-SS men did the shooting.

[10] The killing activities of both the Einsatzgruppen detachments and the Police Regiment South slowed down the farther the Wehrmacht advanced, as more Jews were able to escape east and the density of the pre-war Jewish population was lower in Eastern Ukraine.

[13] While the activities of the Police Regiment South, the Einsatzgruppen detachment, and the 1st SS Brigade progressed, the reports by the murder squads were being intercepted and decoded by MI6, the British intelligence service.

As part of Ultra, a British signals intelligence program, the codebreaking facilities at Bletchley Park decoded and analysed the messages.

Apparently angered by the scope of the atrocities, Churchill delivered a speech over the radio on August 24, where he stated:[14] Whole districts are being exterminated... Scores of thousands of executions are perpetrated by the German police troops upon the Soviet patriots defending their native soil.

On 12 September, the Order Police changed their cipher; the following day, SS officials were instructed to stop transmitting their reports over the radio.