Clinical use of methotrexate (MTX) in cancer chemotherapy is limited due to its side effects, notably associated with increased oxidative stress and hepatotoxicity. Zobo is an aqueous extract of Hibiscus sabdariffa known to contain natural antioxidants. The present study investigated the hepatoprotective effect of zobo drink (ZD) on MTX-induced oxidative stress and hepatotoxicity in rats. Rats randomized to control group received distilled water orally; MTX group received intraperitoneal (ip) injection of MTX (20 mg/kg) on day 11; ZD + MTX group was administered ZD (10 ml/kg) for 14 days and was injected with MTX on day 11. Three days after MTX injection, enzyme markers of liver injury, protein profile, and bilirubin were assessed in serum. Hepatic activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) and level of lipid peroxidation were estimated. Histopathological changes were carried out on the liver tissue. MTX induced prominent oxidative hepatotoxicity demonstrated by significant (p < .01) increases in serum liver enzymes and bilirubin, while protein profile was markedly reduced (p < .05). Hepatic activities of SOD, CAT, and GPx considerably decreased, whereas lipid peroxidation increased significantly in the MTX group compared to control. By contrast, ZD administration attenuated and restored the markers of liver injury, hepatic antioxidant enzymes, and lipid peroxidation near to control, while histopathological alterations were ameliorated compared to the MTX group. ZD affords superior protection against MTX-induced oxidative hepatotoxicity via improvement in antioxidant defense systems in rats. ZD could be a potent natural product against hepatotoxicity associated with MTX chemotherapy in cancer patients.