She carried out two patrols, but did not sink any ships.
The boat was sunk in April 1945 by a Polish aircraft in the Atlantic Ocean.
German Type VIIC/41 submarines were preceded by the heavier Type VIIC submarines. U-321 had a displacement of 759 tonnes (747 long tons) when at the surface and 860 tonnes (850 long tons) while submerged. She had a total length of 67.10 m (220 ft 2 in), a pressure hull length of 50.50 m (165 ft 8 in), a beam of 6.20 m (20 ft 4 in), a height of 9.60 m (31 ft 6 in), and a draught of 4.74 m (15 ft 7 in). The submarine was powered by two Germaniawerft F46 four-stroke, six-cylinder superchargeddiesel engines producing a total of 2,800 to 3,200 metric horsepower (2,060 to 2,350 kW; 2,760 to 3,160 shp) for use while surfaced, two Garbe, Lahmeyer & Co. RP 137/c double-acting electric motors producing a total of 750 metric horsepower (550 kW; 740 shp) for use while submerged. She had two shafts and two 1.23 m (4 ft)propellers. The boat was capable of operating at depths of up to 230 metres (750 ft).
The submarine had a maximum surface speed of 17.7 knots (32.8 km/h; 20.4 mph) and a maximum submerged speed of 7.6 knots (14.1 km/h; 8.7 mph). When submerged, the boat could operate for 80 nautical miles (150 km; 92 mi) at 4 knots (7.4 km/h; 4.6 mph); when surfaced, she could travel 8,500 nautical miles (15,700 km; 9,800 mi) at 10 knots (19 km/h; 12 mph). U-321 was fitted with five 53.3 cm (21 in)torpedo tubes (four fitted at the bow and one at the stern), fourteen torpedoes, one 8.8 cm (3.46 in) SK C/35 naval gun, (220 rounds), one 3.7 cm (1.5 in) Flak M42 and two 2 cm (0.79 in) C/30 anti-aircraft guns. The boat had a complement of between forty-four and sixty.