Objective To investigate the protective effect of melatonin on rat liver injury induced by bile duct ligation. Methods Sixty-four rats were randomly divided into four groups：control group （CN group， n=16）， shamoperation group （SO group， n=16）， bile duct ligation group （BDL group， n=16）， and bile duct ligation with melatonin injection （BDL＋MT group， n=16）. The model of obstructive jaundice was done by ligation of the common bile duct. Melatonin was injected daily （0.5 mg/kg） via peritoneal cavity from 1 d before the operation to 7 d following oper-ation. On day 4 and 8 after the ligation， the plasma levels of total bilirubin （TBIL）， alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， gamma glutamyl transferase （GGT）， and alkaline phosphatase （AKP） were measured by routine methods. Malonaldehyde （MDA）， superoxide dismutase （SOD）， glutathione （GSH）， catalase （CAT）， and glutathione peroxidase （GSH-Px） in the liver tissue were determined by spectrophtometry， too. Hepatocytes apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated deoxynuridine triphosphate nick-end labeling （TUNEL） assay. Results Compared with the CN group and SO group， the levels of TBIL， ALT， AST， GGT， and AKP in the plasma， the content of MDA in the liver tissue， and the apoptosis index （AI） in the hepatocyte markedly increased （P＜0.05， P＜0.01）， the content of GSH and the activities of SOD， CAT， and GSH-Px in the liver tissue markedly decreased（P＜0.01） in the BDL group. Compared with the BDL group， the levels of TBIL， ALT， AST， GGT， and AKP in the plasma， the content of MDA in the liver tissue， and the AI in the hepatocyte markedly decreased （P＜0.01）， the content of GSH and the activities of SOD， CAT， and GSH-Px in the liver tissue markedly increased （P＜0.01） in the BDL＋MT group. In the BDL group， the level of MDA in the liver tissue and the levels of TBIL， ALT， AST， GGT， and AKP were positively correlated （P＜0.01）， the content of GSH and the activities of SOD， CAT， and GSH-Px in the liver tissue and TBIL， ALT， AST， GGT， and AKP were negatively correlated （P＜0.01）. The level of MDA in the liver tissue and AI in the hepatocyte was positively correlated （P＜0.01）. The content of GSH and the activities of SOD， CAT， and GSH-Px in the liver tissue and AI were negatively correlated （P＜0.01）. Conclusions The participation of free radical of oxygen in the pathogenesis and severity of cholestasis produced by the acute obstruction of the extra-hepatic biliary duct is likely. The result of the present study indicates that melatonin exerts a protective effect on cholestatic liver injury in rats with BDL. The mechanism of melatonin’s protection on hepatocyte may be through its antioxidant action and by inhibiting hepatocyte apoptosis.