Objective To investigate the mechanism of α- naphthyl isothiocyanate( ANIT) inducing cholestatic hepatitis. Methods A total of 60 healthy male Sprague- Dawley rats were randomly divided into normal control group( N group with 30 rats) and model group( ANIT group with 30 rats),and each group was further divided into three subgroups according to different time phases after gavage( at 24,48,and 72 hours after gavage),with 10 rats in each group. The serum levels of alanine aminotransferase( ALT),aspartate aminotransferase( AST),gamma- glutamyl transpeptidase( GGT),and total bilirubin( TBil) were measured,and the level of malondialdehyde( MDA) in the liver was measured. A light microscope was used to observe the pathological changes in the liver,and a transmission electron microscope was used to observe the changes in the ultrastructure of the bile canaliculus. A confocal laser scanning microscope was used to analyze the mean fluorescence intensity of phalloidine labeled by fluorescein isothiocyanate in order to determine the change in the level of fibrous actin( F- actin). Results After the administration of ANIT by gavage,the model group showed significant increases in the serum levels of ALT,AST,GGT,and TBil and the level of MDA in liver tissue compared with the normal control group. The model group also showed damage in the structure of the hepatic lobules,hyaline degeneration of hepatocytes,spotted,patchy,and focal necrotic lesions,and neutrophil infiltration mostly confined to the hepatocytes around the bile duct,proliferation of biliary epithelial and fibrous tissues,widened perisinusoidal spaces,dilation of the bile canaliculi,extensive shedding of microvilli,and formation of bile thrombi in the bile capillaries,as well as a low fluorescence intensity of F- actin. The above changes were the most obvious at 48 hours; recovery began at 72 hours,but significant differences were still seen between the two groups. Conclusion ANIT can cause lipid peroxidation in liver tissue and lead to degeneration and necrosis of liver cells,damage of microvilli of the bile canaliculi,and formation of bile thrombi,as well as reduction in the expression of F- actin in the bile canaliculi. Therefore,it affects the function of the liver to secrete bile and causes cholestatic hepatitis.