As was the case with many of the other experts, he got off to a slow start in combat. On 31 May 1941, he bailed out of his Bf 109 F-2 because of engine trouble, and he landed at Spikeroog, only lightly injured. Kittel scored quickly and shot down an SB-2 and Yak-1 on his first mission, on the opening day of Operation Barbarossa, June 22, 1941. By the end of 1941, he had 17 victories. On February 19, 1943, Feldwebel Kittel scored his 39th victory, which was also JG 54's 4,000th of the war. JG 54 Kommodore Hannes Trautloft congratulated Kittel and said the following: "I have instructed that you're no longer to be assigned as wingman. Instead you're to be sent on 'freie Jagd' on your own whenever there's an opportunity."

He received the Knight's Cross of the Iron Cross (Ritterkreuz des Eisernen Kreuzes) on 29 October 1943[1] upon achieving his 123rd victory. He received the "Oak Leaves" to go with it in April 1944.

From November 1943 through January 1944, he was Instructor of the EJGr.Ost, in Biarritz, France. On the 31 December 1943, Kittel started to attack an American bomber formation, but he did not get involved in a prolonged dogfight. In March 1944, Kittel returned to JG 54 on the Russian Front.

He earned his "Swords" (Schwerter) on 25 November 1944[1] after achieving his 239th victory.

Kittel was killed on 14 February 1945 (his 583rd mission), over Courland, by an Ilyushin Il-2 pilot.

Otto Kittel flew 583 combat missions and scored 267 kills, 94 of them Il-2's, making him the fourth highest scoring ace of all time, the highest scoring in JG 54 and the highest scoring Focke-Wulf 190 ace. He started the war as Unteroffizier and died as a Staffelkapitän of 2./JG 54.