Calculus Bovis Sativus (also referred to as in vitro Cultured Calculus Bovis), an artificial substitute of natural Calculus Bovis (Niuhuang in Chinese, a traditional Chinese medicine), has been widely used to relieve fever, diminish inflammation and normalize gallbladder function in the last decade. This study aims to investigate the effects and possible mechanisms of Calculus Bovis Sativus on α-naphthylisothiocyanate (ANIT)-induced intrahepatic cholestasis in rats. Calculus Bovis Sativus (50, 100 and 200 mg/kg per day) was intragastrically (i.g.) given to experimental rats for seven consecutive days. A single dose of ANIT (100 mg/kg i.g.) was given to rats on the fifth day to induce intrahepatic cholestasis. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkalinephosphatase (ALP) and total bilirubin (TBIL) were determined through biochemical methods. The bile duct was cannulated with a PE 10 polyethylene tube to collect bile for two hours and bile flow was calculated by the weight of each specimen. Moreover, the mechanism of Calculus Bovis Sativus was elucidated by determining liver malondialdehyde (MDA) content and superoxide dismutase (SOD) activity. The biochemical observations were supplemented by histopathological examinations. Our results showed that Calculus Bovis Sativus (50, 100 and 200 mg/kg) significantly prevented ANIT-induced changes in bile flow and serum levels of ALT, AST, ALP and TBIL. Furthermore, Calculus Bovis Sativus (50, 100 and 200 mg/kg) significantly reduced the elevated hepatic MDA content induced by ANIT and increased the hepatic SOD activity suppressed by ANIT. Accordingly, histopathology of the liver tissue showed that pathological injuries were relieved after Calculus Bovis Sativus (50, 100 and 200 mg/kg) pretreatment. In conclusion, Calculus Bovis Sativus exerted a protective effect on ANIT-induced intrahepatic cholestasis in rats, which may result from the attenuated oxidative damage in liver tissues.