Hepatotoxicity is one of the major side effects of methotrexate (MTX), which restricts the clinical use of this drug. Berberine (BBR) is a natural compound with multiple pharmacological activities such as antioxidant, antiapoptotic and anti-inflammatory effects. In this study, the effect of BBR on MTX-induced hepatotoxicity was studied. A total number of 28 male Wistar rats were randomly divided into four experimental groups. Rats were pretreated with BBR orally with dose of 100mg/kg for 10 consecutive days and MTX (20mg/kg, intraperitoneally) was administrated on the 9th day. Then on day 11, blood samples were collected to determine serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP). The extracted livers were used for histological examination, biochemical assays and real time PCR studies. Malondialdehyde (MDA), glutathione (GSH), protein carbonyl (PC), nitric oxide (NO) levels, catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and myeloperoxidase (MPO) activities were assessed in hepatic tissue. In addition, the expression of SOD and PGx was measured using real-time PCR method in hepatic tissue. Results showed that MTX administration significantly increases AST, ALT and ALP levels (all p<0.001). It also, increases MDA, PC, NO levels and MPO activity (p<0.001, p<0.01, p<0.05 and p<0.01 respectively). Moreover, MTX decreases hepatic GSH level, SOD, GPx and CAT activities (all p<0.001). Pre-treatment with BBR for 10days prevented some of these changes. Serum levels of AST and ALT decreased (all p<0.001). Hepatic MDA level decreased (p<0.001) and GSH level as well as GPx activity increased (p<0.05 and p<0.01 respectively). Our results indicated that BBR might be useful for prevention of the hepatotoxicity induced by MTX via ameliorative effects on biochemical and oxidative stress indices.