Methotrexate (MTX) is an anti-neoplastic drug that is widely used in the treatment of several diseases such as lymphoblastic leukemia, lymphoma, and autoimmune diseases, and has been concomitant with mild or severe liver and intestinal toxicity. One of the most attractive approaches to prevent toxicity of different organs involves the use of natural antioxidants. Thus, the present investigation was undertaken to study the possible protective effect of wheat germ oil (WGO) against single dose of methotrexate induced oxidative stress in hepatic and intestinal tissue. The oxidative stress was induced using single intraperitoneal injection of methotrexate (20 mg/kg body weight), while the protecting effect was investigated by oral administration of WGO (1400 mg/kg body weight), for six and three consecutive days before and after MTX injection respectively. MTX has revealed significant depletion of some oxidative biomarkers and an elevation in the activities of liver enzymes, Lactate dehydrogenase (LDH), intestinal malondialdehyde (MDA), lipid profile, and remarkable intestinal DNA damage, as well as abnormal architecture of mucosa. The present findings confirmed that WGO has significant preventive effect against liver injury and intestinal oxidative stress induced by MTX through improving the liver enzymes levels, enhancing the intestinal antioxidant defense status, reduction of lipid peroxidation level, and protection against different pathological changes. Taken together, it's then recommended to give WGO to individuals who are treated with MTX, although more pharmacological and molecular assays are necessary to elucidate its protective mechanisms