Methotrexate (MTX), a folate antagonist agent, is mainly used in treatment of malignant tumors and autoimmune diseases. The present study was undertaken to determine whether antioxidant vitamin (vitamin A) could ameliorate methotrexateinduced oxidative stress in male rabbits. Twenty male rabbits were randomly assigned into four groups. Group 1: control group, Group 2: MTX-treated group (received 20 mg/kg MTX intraperitoneally), Group 3: Vit. A treated group received 5000 IU Vit. A orally) and Group 4: MTX+ Vit. A treated group received MTX 20 mg/kg plus 5000 IU vit. A). After 4 weeks of treatment, blood samples were collected by cardiac puncture to determine the serum malondialdehyde (MDA), as a good indicator for lipid peroxidation and oxidative stress. The results showed that there was a significant increase (p< 0.05) in serum MDA level in MTX-treated group compared with control group. Also, the results revealed that there was a significant decrease (p< 0.05) in serum MDA level in MTX+ Vit. A treated group in comparison with MTX-treated group. The present study suggests that the administration of vit. A with MTX is associated with reduction in oxidative stress, and therapeutic role in reduced toxicity of MTX.