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The large number of defects noted during flight testing of the MiG-1 forced Mikoyan and Gurevich to make a number of modifications to the design. Testing was done on a full-size aircraft in the T-1 wind tunnel belonging to the Central Aero and Hydrodynamics Institute (TsAGI) to evaluate the problems and their proposed solutions. The first aircraft to see all of these changes applied was the fourth prototype of the I-200. It first flew on 29 October 1940 and was approved for production after passing its State acceptance trials. The first MiG-3 as the improved aircraft was named on 9 December, was completed on 20 December 1940 and another 20 were delivered by the end of the year.

MiG-3s were delivered to front-line fighter regiments beginning in the spring of 1941 and proved to be a handful for pilots accustomed to the lower-performance and docile Polikarpov I-152 and I-153 biplanes and the Polikarpov I-16 monoplane. Even after the extensive modifications made to the MiG-3 in comparison to the MiG-1 it was still tricky and demanding to fly. Many fighter regiments were not diligent in training their pilots to handle the MiG as it flew very differently than the older fighters and the rapid pace of deliveries aggravated things so that many units had more MiGs than they had trained pilots to fly them by the time of Operation Barbarossa. On 1 June 1941, 1,029 MIG-3s were on strength, but there were only 494 trained pilots. In contrast to the untrained pilots of the 31st Fighter Regiment those of the 4th Fighter Regiment were able to claim three German high-altitude reconnaissance aircraft shot down before war broke out in June 1941. However high-altitude combat of this sort was to prove to be uncommon on the Eastern Front where most air-to-air engagements were at altitudes well below 5,000 metres (16,000 ft). At these sorts of altitudes the MiG-3 was outclassed by the Bf 109 in all respects, but also by other modern Soviet fighters like the Yakovlev Yak-1. Furthermore, the shortage of ground-attack aircraft in 1941 forced it into that role as well, for which it was totally unsuited. Pilot Alexander E. Shvarev recalled: "The Mig was perfect at altitudes of 4,000 m and above. But at lower altitudes it was, as they say, 'a cow'. That was the first weakness. The second was its armament: weapons failure dogged this aircraft. The third weakness was its gunsights, which were inaccurate: that's why we closed in as much as we could and fired point blank."

On 22 June 1941, most MiG-3s and MiG-1s were in the border military districts of the Soviet Union. The Leningrad Military District had 164, 135 were in the Baltic Military District, 233 in the Western Special Military District, 190 in the Kiev Military District and 195 in the Odessa Military District for a total of 917 on hand, of which only 81 were non-operational. An additional 64 MiGs were assigned to Naval Aviation, 38 in the Air Force of the Baltic Fleet and 26 in the Air Force of the Black Sea Fleet.

The 4th and 55th Fighter Regiments had most of the MiG-3s assigned to the Odessa Military District and their experiences on the first day of the war may be taken as typical. The 4th, an experienced unit, shot down a Romanian Bristol Blenheim reconnaissance bomber, confirmed by post-war research, and lost one aircraft which crashed into an obstacle on take-off. The 55th was much less experienced with the MiG-3 and claimed three aircraft shot down, although recent research confirms only one German Henschel Hs 126 was 40% damaged, and suffered three pilots killed and nine aircraft lost. The most unusual case was the pair of MiG-3s dispatched from the 55th on a reconnaissance mission to Ploiesti that failed to properly calculate their fuel consumption and both were forced to land when they ran out of fuel.

Most of the MiG-3s assigned to the interior military districts were transferred to the PVO where their lack of performance at low altitudes was not so important. On 10 July 299 were assigned to the PVO, the bulk of them belonging to the 6th PVO Corps at Moscow, while only 293 remained with the VVS, and 60 with the Naval Air Forces, a total of only 652 despite deliveries of several hundred aircraft. By 1 October, on the eve of the German offensive towards Moscow codenamed Operation Typhoon, only 257 were assigned to VVS units, 209 to the PVO, and 46 to the Navy, a total of only 512, a decrease of 140 fighters since 10 July, despite deliveries of over a thousand aircraft in the intervening period. By 5 December, the start of the Soviet counter-offensive that drove the Germans back from the gates of Moscow, the Navy had 33 MiGs on hand, the VVS 210, and the PVO 309. This was a total of 552, an increase of only 40 aircraft from 1 October.

Over the winter of 1941-42 the Soviets transferred all of the remaining MiG-3s to the Navy and PVO so that on 1 May 1942 none were left on strength with the VVS. By 1 May 1942, Naval Aviation had 37 MiGs on strength, while the PVO had 323 on hand on 10 May. By 1 June 1944, the Navy had transferred all its aircraft to the PVO, which reported only 17 on its own strength, and all of those were gone by 1 January 1945. Undoubtedly more remained in training units and the like, but none were assigned to combat units by then.

Even with the MiG-3's limitations, Aleksandr Pokryshkin, the third-leading Soviet, and Allied, ace of the war, with 53 official air victories (plus six shared), recorded a number of those victories while flying a MiG-3 at the beginning of the war. He later recalled:

“Its designers rarely succeeded in matching both the fighter's flight characteristics with its firepower… the operational advantage of the MiG-3 seemed to be obscured by its certain defects. However, these advantages could undoubtedly be exploited by a pilot able to discover them”.

The MiG-3's top speed of 640 km/h (398 mph) at 7,200 metres (23,622 ft) was faster than the 615 km/h (382 mph) of the German Messerschmitt Bf 109F-2 in service at the beginning of 1941 and the British Supermarine Spitfire V's 603 km/h (375 mph). At lower altitudes the MiG's speed advantage disappeared as its maximum speed at sea level was only 505 km/h (314 mph) while the Bf 109F-2 could do 515 km/h (320 mph).Unfortunately for the MiG-3 and its pilots, aerial combat over the Eastern Front generally took place at low and medium altitudes where it had no speed advantage.

The MiG's loaded weight of 3,350 kg (7,385 lb) was greater than the Bf 109F-2's 2,728 kg (6,014 lb) and it was less maneuverable in the horizontal plane than the Bf 109 due to its higher wing loading. This lack of maneuverability was exacerbated by the MiG-3's poor climb performance, its instability at high speeds (which can make aerial gunnery difficult due to the point of aim "wandering" and requiring constant pilot input to remain on target), and its underpowered armament.

The MiG-3's standard armament was one 12.7 mm (0.50 in) UBS machine gun and two 7.62 mm (0.30 in) ShKAS machine guns, all mounted in the engine cowling and synchronized to fire through the propeller arc. In contrast, most versions of the German Messerschmitt Bf 109 that it encountered had one 20 mm (0.79 in) cannon and two 7.92 mm (0.31 in) machine guns (although the Bf 109F used during Operation Barbarossa had the 15mm MG 151/20, meaning that it was armed little better than the MiG). To remedy this problem, 821 aircraft were built with one 12.7 mm UBK machine gun in a pod under each wing in mid-1941. This lowered its speed by about 20 km/h (12 mph) at all altitudes, which was unpopular with the pilots, some of whom removed the pods. One hundred aircraft were equipped with a pair of UBS machine guns in lieu of the ShKAS weapons. Another 215 aircraft also had just the UBS machine guns but were fitted to carry six RS-82 rockets. A total of 72 aircraft mounted a pair of 20 mm ShVAK cannon. A wide variety of armaments were experimented with by various units at the requests of their pilots or to make up shortages.