Dioscorea bulbifera L. is a medicinal plant. The present study was undertaken to investigate the hepatotoxicity induced by D. bulbifera in mice. Through the acute toxicity of various extracts including the EtOAc fraction (EF) and the non-EtOAc fraction (Non-EF) from ethanol, and the ethanol itself, we found that the EF contains the toxic ingredients of D. bulbifera rhizome. On this basis, to study the hepatotoxicity induced by the toxic ingredients, mice were treated with 0.5% sodium carboxymethyl cellulose (CMC-Na) alone or the EF of D. bulbifera rhizome at doses of 80, 160, 320, and 480 mg/kg once daily i.g. for fourteen consecutive administrations. Serum samples were collected for determination of the biomarkers for liver injury, such as, alanine transaminase (ALT) and aspartate transanimase (AST). Hepatic tissues were used to assay for the level of lipid peroxide (LPO), amounts of antioxidants such as glutathione, and activities of antioxidant-related enzymes for liver oxidative-antioxidative status in mice. The results showed that ALT and AST were significantly elevated after fourteen consecutive administrations of the EF of D. bulbifera rhizome. In addition, the level of LPO increased remarkably, while the glutathione amounts, and the activities of the antioxidant-related and glutathione-related enzymes including superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR) and glutamate-cysteine ligase (GCL) of hepatic tissues all decreased conspicuously, in livers of mice treated with the EF of D. bulbifera rhizome. Taken together, our results indicate that the EF contains the main toxic ingredients of D. bulbifera rhizome, and the mechanism of hepatotoxicity induced by it may be due to liver oxidative stress injury in mice.