Objective: To compare the differences between bile duct ligation (BDL) and α-naphthylisothiocyanate (ANIT)-induced mouse models in cholestatic liver injury experiments, and to explore the applicability of both models in such studies. Methods: C57BL/6 male mice were divided into control, sham surgery (Sham), BDL, BDL+SCFA, ANIT, and ANIT+SCFA groups. The BDL and BDL+SCFA groups underwent BDL surgery, while the ANIT and ANIT+SCFA groups were administered ANIT. The BDL+SCFA and ANIT+SCFA groups received water containing short-chain fatty acids (SCFAs) for 14 days. Samples were collected for liver histology, liver function serum biomarker analysis, and inflammatory cytokine detection. Statistical analysis was performed on the data. Results: Mice in the BDL group developed noticeable jaundice and continuous weight loss at one week, with severe jaundice by two weeks. Mice in the ANIT group exhibited mild jaundice early on, which gradually worsened over time, with mild early weight loss and no significant later changes. Gross liver appearance and histopathological analysis revealed common characteristics between the two models, including partial liver fibrosis, hepatocellular necrosis, portal tract fibrosis, inflammatory cell infiltration, and bile duct proliferation. However, these changes were more prominent and occurred earlier in the BDL group, indicating more severe cholestatic liver injury. In contrast, the ANIT group showed milder early changes that progressively worsened over time. Serum biomarkers revealed significant increases in liver transaminase levels in both groups. Early increases in alkaline phosphatase (ALP) and total bilirubin (TBIL) levels were observed in the BDL group, while the ANIT group showed mild early increases, which gradually worsened over time. Serum inflammatory cytokine levels were significantly elevated early in the BDL group, with gradual elevation in the ANIT group. After two weeks of SCFA treatment, there were no significant improvements in jaundice, weight loss, liver serum biomarkers, or inflammatory markers in the BDL group, but significant improvements were observed in the ANIT group, including reduced jaundice, weight loss, liver serum biomarkers, and inflammatory markers. Conclusion: The BDL model has a rapid onset and severe liver injury, making it suitable for studying acute cholestatic liver injury, whereas the ANIT model has a slow onset with progressively worsening liver injury, making it more appropriate for modeling chronic cholestatic liver injury.