This study aimed to investigate the hepatoprotective and antioxidant effect of pumpkin seed oil (PSO) against Carbon tetra chloride (CCl4) induced hepatotoxicity. For this purpose, 40 male albino rats were divided into four groups. Group 1 was maintained as normal control group, were given the vehicle. Group 2 was administered with pumpkin seed oil alone at a dose of 300 mg/kg. b.w /day orally. Group 3 received CCl4 alone at a dose of 2.5 ml/kg b.w. (30%) CCl4 / corn oil intraperitoneally after 2 weeks and after 3 weeks of the experiment. Group 4 was given both 300 mg/kg. b.w./day pumpkin seed oil for 3 weeks and 2.5 ml CCl4 as in (group 3). 48 hours after the 2nd CCl4 injection blood samples and liver tissues were collected for biochemical, histopathological and immunohistochemical examinations. In CCl4 group, ALT, AST, T. bilirubin in serum significantly increased while T. protein and albumin significantly reduced. Hepatic MDA levels increased and antioxidant enzymes (SOD & CAT) were decreased. Group 4 revealed significant decrease of the hepatic damage, ALT, AST, T. bilirubin while T. protein and albumin significantly increased. Hepatic MDA levels decreased and the efficiency of antioxidant enzymes (SOD & CAT) was improved when compared to CCl4 group. Histopatho‌logical examinations further confirmed that the PSO could protect the liver from CCl4-induced damage. Moreover, immunohistochemistry staining demonstrated that pretreatment with PSO significantly inhibited the elevation of iNOS in CCl4-treated rats. It is concluded that pumpkin seed oil has hepatoprotective and antioxidant effects.