Background: Methotrexate (MTX) is one of the most commonly used chemotherapeutic agents in the treatment of cancer patients, however its therapeutic use is limited due to dose dependent hepatotoxicity caused by oxidative damage. Biochanin A (BCA), a naturally occurring dietary isoflavone, has strong cytoprotective, anti-inflammatory, and antioxidant properties. Although protective actions of BCA against various chemical-induced hepatotoxicity including that of carbon tetrachloride and arsenic, none of the studies was made on MTX-induced acute liver injury. Methods: Wistar rats were separated into four groups; Control, MTX, Control+BCA, MTX+BCA. BCA (50 mg/kg/day) or vehicle (dimethyl sulfoxide; 1 mL/kg/day) with the same volume was intraperitoneally injected for 5 days. At sixth day, a single dose of MTX (20 mg/kg) was injected to rats. Twenty-four h after the MTX administration, rats were sacrificed and then blood and liver samples were obtained for biochemical and histopathologic analyses. Results: MTX increased the serum AST, ALT, and LDH levels. Furthermore, malondialdehyde (an indicator of oxidative injury) and myeloperoxidase (an indicator of neutrophil infiltration) levels increased, while total glutathione levels (an indicator of antioxidant status) decreased in liver. MTX-induced hepatotoxicity was also observed histopathologically. BCA substantially improved these alterations induced by MTX administration. Conclusion: These results indicate that BCA may be useful in preventing the MTX-induced acute liver injury due to its antioxidant and anti-inflammatory properties.