Defensive armament
The Avro Lancaster was initially equipped with four Nash & Thomson Frazer Nash hydraulically operated turrets mounted in the nose, tail, mid-upper and underside. The original tail turret was equipped with four Browning .303 Mark II machine guns and all other turrets with two such machine guns.

Nose turret
Only the FN-5A nose turret which was similar to the FN-5 used on the preceding Avro Manchester, the Vickers Wellington and the Short Stirling remained unchanged during the life of the design, except in instances where it was removed entirely.

Ventral turret
The ventral (underside) FN-64 turret quickly proved to be dead weight, being both difficult to sight because it relied on a periscope which limited the gunner's view to a 20 degree arc, and too slow to keep a target within its sights. Aside from early B Is and the prototype B IIs, the FN-64 was almost never used. When the Luftwaffe began using Schräge Musik to make attacks from below in the winter of 1943/1944, modifications were made, including downward observation blisters mounted behind the bomb aimer's blister and official and unofficial mounts for .50 in (12.7 mm) machine guns or even 20 mm cannon, firing through the ventral holes of the removed FN-64. The fitting of these guns was hampered as the same ventral position was used for mounting the H2S blister, which limited installations to those aircraft fitted with bulged bomb bays which interfered with the H2S.

Mid-upper turret
The mid-upper (dorsal or top turret) was an FN-50 on early examples and the very similar FN-150 with improved sights and controls on later examples. On all but the earliest examples this turret was surrounded by a coaming which provided a track for a cam operated interruptor device which prevented the gunner from shooting the tail of his own aircraft. The Mk.VII and late Mk.X Lancasters used the heavier electrically-controlled Martin 250 CE 23A turret equipped with two .50 inch machine guns which was mounted further forward to preserve the aircraft's longitudinal balance, and because it had an internal mechanism to prevent firing on the aircraft itself, it did not require a coaming. Other experimental turrets were tried out, including the FN-79 and the Boulton-Paul Type H barbette system.

Tail turret

The tail turret was the most important defensive position and carried the heaviest armament. Despite this, the turrets used, starting with the FN-20, were never entirely satisfactory and numerous designs were tried. The FN-20 was replaced by the very similar FN-120 which used an improved gyroscopic gun sight (GGS). Gunners using both the FN-20 and 120 removed perspex and armour from the turret to improve visibility, but trials by the RAF showed that a Mosquito night fighter was still able to get within a very short distance of the tail gunner without being spotted, confirming what the Luftwaffe had already realised. The Rose turret attempted to improve on the FN turrets by being completely open to the rear (improving visibility and allowing easier emergency egress) and by being fitted with two .50 inch machine guns and was installed in a small number of Lancasters but never became common. Ultimately radar, rather than improved visibility, made the turret more effective. The FN-121 was the Automatic Gun Laying Turret (AGLT), an FN-120 fitted with Village Inn gun-laying radar. Aircraft fitted with Village Inn were used as bait, flying behind the main formations to confront the night fighters that
Flight -Sergeant J Morgan, the rear gunner of an Avro Lancaster of No. 630 Squadron RAF at East Kirkby, Lincolnshire, checks his guns in the Nash & Thompson FN120 tail turret before taking off on a night raid on the marshalling yards at Juvisy-sur-Orge, France.
followed the formations and shot down stragglers. This significantly reduced operational losses; and gun-laying radar was added to the last versions of the turret. Before the end of the war Lancasters built in the UK standardised on the FN-82 fitted with two .50 inch machine guns and fitted with gun-laying radar as production allowed, which was also used on early models of the Avro Lincoln. The disadvantage of all radar and radio transmitting systems is that attacking forces can locate aircraft by picking up transmissions.

Later in the war Freeman Dyson made a case for removing all the Lancaster's defensive armament, arguing it would reduce the loss rate by increasing the Lancaster's speed by up to 50 mph (assuming the bomb load was not increased at the same time), and thus make it harder to shoot down. This became even more important when Dyson and Mike O'Loughlin concluded that some of the German night fighters were using Schräge Musik upward firing guns, as the Lancaster had no ventral gun turret to defend itself, although any defence would depend on the crew detecting the attack from underneath. Dyson considered that the modification would be justified even if the aircraft loss rate was unchanged, as two defensive air gunners would not be required, reducing human losses. The case for speed over defensive armament was supported by the Mosquito, whose loss rates were far lower than the Lancaster's. As an example, during the Battle of Berlin (18 November 1943 to 30 March 1944) the average loss rate of the heavy bombers (overwhelmingly Lancasters) was 5.1%, whereas for Mosquitoes it was 0.5%, though a speed-optimised Lancaster would still be up to 50 mph slower than a Mosquito and unlikely to match its low loss rates.

An important feature of the Lancaster was its unobstructed 33 ft (10 m) long bomb bay. At first, the heaviest bomb carried was the 4,000 lb (1,800 kg) high capacity HC "Cookie". Bulged doors were added to 30% of B Is to allow the aircraft to carry 8,000 lb (3,600 kg) and later 12,000 lb (5,400 kg) "Cookies". The Lancaster also carried a variety of smaller weapons, including the Small Bomb Container (SBC) which held 236 4 lb (1.8 kg) or 24 30 lb (14 kg) incendiary and explosive incendiary bomblets; 500 lb (230 kg) and 1,000 lb (450 kg) General Purpose High Explosive (GP/HE) bombs (these came in a variety of designs); 1,850 lb (840 kg) parachute deployed magnetic or acoustic mines, or 2,000 lb (910 kg) armour-piercing (AP) bombs; 250 lb (110 kg) Semi-Armour-Piercing (SAP) bombs, used up to 1942 against submarines; post 1942: 250 lb (110 kg) or 500 lb (230 kg) anti-submarine depth charges.

In 1943 617 Squadron was created to carry out Operation Chastise, the raid against the Ruhr dams. This unit was equipped with B.III (Specials), officially designated the "Type 464 (Provisioning)", modified to carry the 9,250 lb (4,200 kg) "Upkeep" bouncing bomb. The bomb bay doors were removed and the ends of the bomb bay were covered with fairings. "Upkeep" was suspended on laterally pivoted, vee-shaped struts which sprang apart beamwise when the bomb-release button was pressed. A drive belt and pulley to rotate the bomb at 500 rpm was mounted on the starboard strut and driven by a hydraulic motor housed in the forward fairing. The mid-upper turret was removed and a more bulbous bomb aimer's blister was fitted; this, as "Mod. 780", later becoming standard on all Lancasters, while the bombsight was replaced by a simple aiming device. Two Aldis lights were fitted in the rear bomb bay fairing; the optimum height for dropping "Upkeep" was 60 ft and, when shone on the relatively smooth waters of the dam's reservoirs, the light beams converged into a single spot when the Lancaster was flying at the correct height.

Towards the end of the war, attacking special and hardened targets, other variants of B I Specials were modified to carry the 21 ft (6.4 m) long 12,000 lb (5,400 kg) "Tallboy" or 25.5 ft (7.8 m) long 22,000 lb (10,000 kg) "Grand Slam" "earthquake" bombs. Aircraft intended to carry the "Grand Slam" required extensive modifications. These included the removal of the dorsal turret and of two guns from the rear turret, removal of the cockpit armour plating (the pilot's seatback), and installation of Rolls-Royce Merlin Mk 24 engines for better take-off performance. The bomb bay doors were removed and the rear end of the bomb bay cut away to clear the tail of the bomb. Later the nose turret was also removed to further improve performance. A strengthened undercarriage and stronger mainwheels, later used by the Avro Lincoln, were fitted.