This study aimed to investigate the protective effect of wedelolactone on D-galactosamine-induced acute liver injury (ALI) in mice. Fifty mice were divided into normal, model and 5, 10, and 20 mg/kg wedelolactone groups, 10 mice for each group. The latter three groups were treated with 5, 10, and 20 mg/kg by intragastric administration, respectively, for consecutive days. Then, the D-galactosamine-induced ALI model was prepared in the latter four groups. Results showed that the wedelolactone pre-treatment could decrease the liver index, serum ALT, and AST levels, and liver tissue xanthine oxidase, malon-dialdehyde, nitric oxide, inducible nitric oxide synthetase (iNOS), interferon-gamma and tumor necrosis factor alpha levels, and increase the liver tissue superoxide dismutase, glutathione peroxidase, interleukin 4 and interleukin 10 levels in ALI mice. In conclusion, wedelolactone has protective effect on acute D-galactosamine ALI in mice. The mechanism might be related to its resisting free radical damage and lipid peroxidation, inhibiting iNOS over-expression and regulating Th1/Th2 balance.