Objective: Malathion is widely used as a potent pesticide in many countries and has been shown to produce some adverse health effects via induction of oxidative stress in through the generation of free radicals and alteration of the cellular antioxidant defense system. The aim of this study was to investigate malathion-induced oxidative DNA injury in rat sperm by measuring the activities of plasma peroxynitrite (ONOO–), glutathione peroxidase (GPx), superoxide dismutase (SOD), Mn-SOD, catalase (CAT), and lipid peroxidation (LPO) in rats. Materials and Methods: 12 male rats of 220-230 g were divided into two groups as follows: group 1 received only distilled water, group 2 received malathion (200 mg/kg/day) dissolved in distilled water. The groups were treated for 7 days by intraperitoneal injection. To evaluate sperm chromatin quality and DNA integrity, Aniline blue and Acridine orange staining were done in the groups. Results: In the blood plasma, the LPO, ONOO– and GPx were higher in the malathion group as compared with controls. Also plasma CAT decreased in malathion-treated animals comparing with controls. Malathion did not alter plasma total SOD and Mn-SOD. These changes were associated with significant increase in DNA damage and chromatin abnormality in the cauda epididymal sperm as evidenced by acridine orange and aniline blue staining respectively. Conclusion: The present results highly support the idea that sperm DNA damage of malathion is mediated through oxidative stress.