The role of xanthine oxidase (XO) in the defense response of wheat (Triticum aestivum L.) against the Russian wheat aphid (RWA) (Diuraphis noxia) was studied. Xanthine oxidase catalyzes the oxidation of hypoxanthine to xanthine to uric acid, and oxygen radicals that are formed as a by-product at both of these oxidation steps may participate in plant defense reactions. A resistant wheat cultivar (Tugela DN), and a close isogenic susceptible cultivar (Tugela), were infested with 20–30 aphids per plant before inhibiting xanthine oxidase by adding allopurinol as a soil drench to the resistant plants. Increases in H2O2 content were detected early after infestation in the resistant wheat, and the downstream defense related responses, chitinase and peroxidase activities, were selectively induced by RWA infestation. These downstream defense responses were substantially inhibited in allopurinol treated-resistant wheat, presumably as a response to inhibition of XO, and superoxide dismutase (SOD). We conclude that the production of active oxygen species through the XO system plays an important role in the induction of defense reactions in wheat against the RWA.