Methotrexate (Mtx) is an effective chemotherapeutic agent used in various cancer treatments. Gastrointestinal toxicity is the drug's major limiting factor, arising mainly from oxidative damage. It has been proposed that ozone (O(3)) is an activator of antioxidant enzymes. Thus, this study was designed to investigate the efficacy of ozone therapy in the prevention of Mtx-induced intestinal injury in rats. Twenty rats were allocated into three groups: sham, Mtx alone (untreated) and Mtx + O(3) (treated with ozone). Ozone was administered at a dose of 0.72 mg/kg daily via an intraperitoneal route for 15 d. On d 16, Mtx was applied via an intraperitoneal injection at a dose of 6 mg/kg for 5 d. All rats were sacrificed at d 21. Efficacy of the treatment was assessed by measuring the histopathologic injury score (HIS), and biochemically by determining tissue superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in ileum, liver and kidney homogenates. Although two rats (25%) died in the untreated group, all rats in the sham and treatment groups survived the study. The HIS, antioxidant enzyme and MDA levels of the ileal tissue were significantly lower in the ozone treated group than the untreated group (p < 0.05). Although the antioxidant enzyme and MDA levels of liver and kidney were significantly lower in the ozone treated group (p < 0.05), there was no significant change in histopathology (p > 0.05). Thus, ozone preconditioning shows a preventative effect in the ileum by decreasing tissue damage and increasing antioxidant enzyme activity in an experimental model of Mtx-induced intestinal injury.