Chemotherapeutic agent, busulfan, induce oxidative stress as a mechanism to kill cancer cells, however, it may also cause oxidative stress in non-target tissues and thereby lead to normal tissue injury. Milk thistle (Silybum marianum L.) has been used for centuries as a herbal drug. The present study was carried out to investigate the potential effects of wild Silybum marianum seed ethanol extract (SMSEE) intervention on oxidative stress induced by busulfan drug in different organs of Rats. For the study 42 rats were prepared and fed with special ration, then they were divided to 7 groups with 6 rats in each group: group 1, Normal control: healthy rats without intervention; group 2.control SMSEE, 400 mg/kg/day SMSEE; group 3, positive control group received 20 mg/kg/day busulfan, group 4, treated group received 20 mg/kg/day busulfan + 200 mg/kg/day SMSEE; group 5, treated group received 20 mg/kg/day busulfan+ 400 mg/kg/day SMSEE; group 6, treated group received 20 mg/kg/day busulfan + 600 mg/kg/day SMSEE; group 7, treated group received 20 mg/kg/day busulfan + 800 mg/kg/day SMSEE. The amount of oxidative stress parameters (ROS and MDA), glutathione fractions (GSH and GSSG), and antioxidant enzymes (GSH-Px, GSH-Rd, SOD and CAT) in the different tissue extracts (liver, kidneys, heart, spleen, pancreas and testes) were measured. The results of this study showed that after busulfan treatment the levels of ROS and MDA were significantly (p≤0.05) increased and GSH, GSSG, GSH-Px, GSH-Rd, SOD and CAT were decreased in all studied organs. Treatment of busulfan administration rats with SMSEE leads to the opposite direction by different rates. In conclusion, the use of busulfan in rats administration induce oxidative stress in different organs and SMSEE had an preventive role through decreasing the ROS and the lipid peroxidation, and improvement the oxidative defense system. These results provide a basis for the use of Silybum marianum extracts as promising tools in the future for many important nutritional and therapeutic applications