<p>This study aimed to investigate the therapeutic potential and the involvements of PPARα-mediated pathways in the mechanism of San-Huang-Chai-Zhu Formula (SHCZF) in intrahepatic cholestasis (IC). The therapeutic potential of SHCZF was evaluated in alpha-naphthylisothiocyanate (ANIT)-induced IC rat model, with ursodeoxycholic acid (UDCA) used as positive control. Liver pathology was assessed by hematoxylin-eosin staining. Curative effects were evaluated by histopathological staining, and by detecting the levels of bile acid homeostasis-related, glutathione (GSH) homeostasis-related factors and inflammatory markers. GW6471, a PPARα antagonist, was applied to explore the involvements of PPARα-mediated pathway in the mechanism of SHCZF in IC. SHCZF markedly improved the abnormal biochemical parameters, liver lesions, restored bile acid and GSH homeostasis, and reduced inflammation level in ANIT-induced IC rats, similarly to that of UDCA. ANIT-induced IC rats showed significantly decreased PPARα expression and increased p-JNK and p-MKK4 expression. Such changes caused by ANIT could be reversed after treatments with UDCA and/or SHCZF. PPARα antagonist GW6471 treatment reduced PPARα levels and partly counteracted the curative effects of SHCZF. Importantly, GW6471 altered the regulatory effects of SHCZF on MKK4/JNK pathway and bile acid homeostasis. SHCZF improves IC by regulating PPARα-mediated bile acid homeostasis and MKK4/JNK pathway.</p>