From the beginning, the U-2 became the basic Soviet civil and military trainer aircraft, mass-produced in a "Red Flyer" factory near Moscow. It was also used for transport, and as a military liaison aircraft, due to its STOL capabilities. Also from the beginning it was produced in an agricultural aircraft variant, what earned it its nickname Kukuruznik. Although entirely outclassed by contemporary aircraft, the Kukuruznik served extensively on the Eastern Front in World War II, primarily as a liaison, medevac and general supply aircraft. It was especially useful for supplying Soviet partisans behind the front line. Its low cost and easy maintenance led to a production run of over 40,000. Manufacturing of the Po-2 in the USSR ceased in 1949, but until 1959 a number were assembled in Aeroflot repair workshops.
First trials of arming the aircraft with bombs took place in 1941.
During the defence of Odessa, in September 1941, the U-2 was used as a reconnaissance aircraft and as a light, short-range, bomber. The bombs, dropped from a civil aircraft piloted by Pyotr Bevz, were the first to fall on enemy artillery positions. From 1942 it was adapted as a light night ground attack aircraft.
Nikolay Polikarpov supported the project, and under his leadership, the U-2VS (voyskovaya seriya - Military series) was created. This was a light night bomber, fitted with bomb carriers beneath the lower wing, to carry 50 or 100 kg (110 or 220 lbs) bombs up to a total weight of 350 kg (771 lb) and armed with ShKAS or DA machine guns in the observer's cockpit.
Wehrmacht troops nicknamed it Nähmaschine (sewing machine) for its rattling sound and Finnish troops called it Hermosaha (Nerve saw) as the Soviets flew nocturnal missions at low altitudes: the engine had a very peculiar sound, which was described as nerve-wracking, therefore the name. The enemy soon became aware of the threat posed by the U-2, and Luftwaffe pilots were given special instructions for engaging these aircraft. The material effects of these missions may be regarded as insignificant, but the psychological effect on German troops was much more noticeable. They typically attacked by complete surprise in the dead of night, denying German troops sleep and keeping them constantly on their guard, contributing yet further to the already exceptionally high stress of combat on the Eastern front. Their usual tactics involved flying only a few meters above the ground, rising for the final approach, cutting off the engine and making a gliding bombing run, leaving the targeted troops with only the eerie whistling of the wind in the wings' bracing-wires as an indication of the impending attack. Luftwaffe fighters found it extremely hard to shoot down the Kukuruznik because of three main factors: the rudimentary aircraft could take an enormous amount of damage and stay in the air, the pilots used the defensive tactic of flying at treetop level, and the stall speed of both the Messerschmitt Bf 109 and the Focke-Wulf Fw 190 was similar to the Soviet aircraft's maximum cruise speed, making it difficult for the newer aircraft to keep a Po-2 in weapons range for an adequate period of time. The success of the Soviet night harassment units using the Po-2 inspired the Luftwaffe to set up similar Störkampfstaffel "harassment combat squadrons" on the Eastern Front using their own obsolete 1930s-era, open cockpit biplane and parasol monoplane aircraft, eventually building up to larger Nachtschlachtgruppe (night attack group) units of a few squadrons each.
The U-2 was known as the aircraft used by the 588th Night Bomber Regiment, composed of an all-women pilot and ground crew complement. The unit became notorious for daring low-altitude night raids on German rear-area positions. Veteran pilots, Yekaterina Ryabova and Nadezhda Popova on one occasion flew eighteen such missions in a single night. The women pilots observed that the enemy suffered a further degree of demoralization simply due to their antagonists being female. As such, the pilots earned the nickname "Night Witches" (German Nachthexen, Russian ?????? ??????/Nocnye Ved’my). The unit earned numerous Hero of the Soviet Union citations and dozens of Order of the Red Banner medals; most surviving pilots had flown nearly 1,000 combat missions by the end of the war and had taken part in the Battle of Berlin.
The Polish Air Force used these slow and manoeuvrable aircraft for air reconnaissance and COIN operations against UPA bands in mountainous area of Bieszczady. Pilots and navigators were dispatched to look for concentrations of UPA forces and if needed, engage them with machine guns and grenades. On several occasions, the UPA managed to bring down some of the Po-2s, but never captured or used one, as pilots were ordered to burn their aircraft in case of a crash.
North Korean forces used the Po-2 in a similar role in the Korean War. A significant number of Po-2s were fielded by the Korean People's Air Force, inflicting serious damage during night raids on Allied bases. On 28 November, at 0300 hours, a lone Po-2 attacked Pyongyang airfield in northwestern Korea. Concentrating on the 8th Fighter-Bomber Group's parking ramp, the Po-2 dropped a string of fragmentation bombs squarely across the Group's lineup of F-51 Mustangs. Eleven Mustangs were damaged, three so badly that they were destroyed when Pyongyang was abandoned several days later.
On 17 June 1951, at 01:30 hours, Suwon Air Base was bombed by two Po-2s. Each biplane dropped a pair of fragmentation bombs. One scored a hit on the 802nd Engineer Aviation Battalion's motor pool, damaging some equipment. Two bombs burst on the flight line of the 335th Fighter Interceptor Squadron. One F-86A Sabre (FU-334 / 49-1334) was struck on the wing and began burning. The fire took hold, gutting the aircraft. Prompt action by personnel who moved aircraft away from the burning Sabre preventing further loss. Yet eight other Sabres had been damaged in the brief attack, four seriously. One F-86 pilot was among the wounded. The North Koreans subsequently credited Lt. La Woon Yung with this damaging attack.
UN forces named the Po-2's nighttime appearance Bedcheck Charlie and had great difficulty in shooting it down — even though night fighters had radar as standard equipment in the 1950s, the wood-and-fabric-construction of the Po-2 gave only a minimal radar echo, making it hard for an opposing fighter pilot to acquire his target. As Korean war U.S. veteran Leo Fournier remarks about "Bedcheck Charlie" in his memoirs later on: "... no one could get at him. He just flew too low and too slow." On 16 June 1953, a USMC AD-4 from VMC-1 piloted by Major George H. Linnemeier and CWO Vernon S. Kramer shot down a Soviet-built Polikarpov Po-2 biplane, the only documented Skyraider air victory of the war. One Lockheed F-94 Starfire was lost while slowing to 110 mph during an intercept of a Po-2 biplane.