Cholestasis is a clinical disorder defined as an impairment of bile flow, and that leads to toxic bile acid (BA) accumulation in hepatocytes. Here, we investigated the hepatoprotective effect of Yinchenhaotang (YCHT), a well-known formulae for the treatment of jaundice and liver disorders, against the cholestasis using the α-naphthylisothiocyanate (ANIT)-induced cholestasis in male Wistar rats. ANIT feeding induced significant cholestasis with substantially increased intrahepatic retention of hydrophobic BAs. The dynamic changes of serum and liver BAs indicated that YCHT was able to attenuate ANIT-induced BA perturbation, which is consistent with the histopathological findings that YCHT significantly decreased the liver damage. YCHT treatment substantially reduced serum alanine aminotransferase (ALT), alkaline phosphatase (AST), total bilirubin (TBIL) and direct bilirubin (DBIL) with minimal bile duct damage in the ANIT treated rats. Elevated mRNA expression of liver IL-6, IL-17A, IL-17F, TGF-β1, α-SMA, TGR5, NTCP, OATP1a1, and ileum ASBT and decreased liver IL-10, FXR, CAR, VDR, BSEP, MRP2, MRP3, MRP4 was also observed in ANIT-induced cholestasis but were attenuated or normalized by YCHT. Our results demonstrated that the BA profiles were significantly altered with ANIT intervention and YCHT possesses the hepatoprotective potential against cholestatic liver injury induced by hepatotoxin such as ANIT.