Diazinon (DZN) as an organophosphate pesticide may cause oxidative stress in different tissues. Antioxidants increase tissue protection from oxidative stress. The aim of the present study was to investigate prophylactic and therapeutic effects of vitamin C against oxidative stress caused by DZN in various tissues of male Wistar rats. Thirty rats were divided into five groups: control group received corn oil as DZN solvent, DZN group received 100 mg/kg of DZN, C group received 200 mg/kg of vitamin C, C-DZN and DZN-C groups received vitamin C before and after DZN injection. Plasma and various tissues were prepared and evaluated for measurement of the biochemical parameters and oxidative stress biomarkers. Results showed that acute administration of DZN significantly increased superoxide dismutase and glutathione-S-transferase activities and malondialdehyde level in all tissues, catalase (CAT) activity in liver, kidney and heart and some plasma biochemical indices, while it decreased cholinesterase and lactate dehydrogenase activities and glutathione content in all tissues. CAT activity in erythrocytes, brain and spleen was decreased in DZN-exposed rats compared with the control group. Administration of vitamins C in both prophylactic and therapeutic groups ameliorated in these parameters, although all these tests in tissues did not return to the normal level. These data suggest that oxidative stress is an essential mechanism involved in DZN-induced adversity effect, as evidenced by the altered activity of antioxidant enzymes, depleted GSH content and the enhanced membrane lipid peroxidation. Both the prophylactic and therapeutic treatments of rats to vitamin C have beneficial effects against oxidative stress and cholinergic hyperactivity induced by DZN in tissues especially in the brain tissue through free radical scavenging.