The first Harrow was delivered to No. 214 Squadron RAF on 13 January 1937, with all 100 delivered by the end of the year, with five bomber squadrons of the RAF being equipped with the Harrow. The Fleet Air Arm ordered 100 Harrows but Handley Page lacked the production capacity to supply them. Despite being fitted with cabin heating by steam boilers using exhaust heat, the Harrow gained a reputation of being a cold and draughty aircraft, owing to the turret design. As the delivery of more modern bombers increased, the Harrow was phased out as a front-line bomber by the end of 1939 but continued to be used as a transport. 271 Squadron was formed on 1 May 1940 with a mixture of Harrows, Bristol Bombays and impressed civil aircraft. While the other aircraft equipping 271 Squadron were replaced by Douglas Dakotas, it retained a flight of Harrows (sometimes nicknamed "Sparrows" due to their new nose fairings to give a more streamlined fuselage) as transports and ambulance aircraft until the end of the Second World War in Europe.
Harrows were used occasionally to operate risky flights between England and Gibraltar, two being lost on this route. Harrows also operated in support of Allied forces, in their advance into north-west Europe, evacuating wounded from the Arnhem operation in September 1944. Seven Harrows were destroyed by a low level attack by Luftwaffe fighters of JG 26 and JG 54 on Evere airfield as part of Operation Bodenplatte, the German attack on Allied airfields in North West Europe on 1 January 1945, leaving only five Harrows, which were eventually retired on 25 May 1945.
The Harrow also served in a novel operational role at the height of the German night Blitz against Britain in the winter of 1940–1941. Six Harrows equipped No. 420 Flight RAF (later No. 93 Squadron RAF) which used lone Harrows to tow Long Aerial Mines (LAM) into the path of enemy bombers. The LAM was an explosive charge on the end of a long cable and the unorthodox tactic was credited with the destruction of six German Bombers or 4–5, depending on the source. The experiment was judged of poor value and the planned deployment of Douglas Havocs in the LAM role was cancelled. Nine Harrows were also used by 782 Naval Air Squadron, Fleet Air Arm as transports. After flight refuelling trials, three Harrows were operated by Flight Refuelling Limited and refuelled Short Empire Flying Boats on transatlantic services, two from Gander, Newfoundland and one based in Foynes, Ireland. In 1940, the two aircraft based at Gander were impressed into service with the Royal Canadian Air Force.