Objective <p>Cholestatic liver injury (CLI) has lacked a potent treatment due to its incompletely understood pathogenesis. Saikosaponin D (SSD), the main active ingredient of the traditional Chinese medicine Bupleuri Radix, has significant anti-inflammatory and hepatoprotective effects and inhibits hepatic fibrosis. This study specifically investigated the protective effects of SSD in CLI and its related mechanisms.</p> Methods <p>In bile duct ligation (BDL)-induced CLI in mice, the protective effect of SSD was assessed by serum biochemical index analysis, HE staining of liver tissue, Sirius red staining, immunohistochemistry and ELISA. Bile acid-induced human HepG2 cell line was taken to study the protective effect and potential mechanism of SSD against CLI in vitro. TLR4/MyD88/NF-κB/NLRP3 pathway-related proteins in mouse liver tissues or HepG2 cells were detected by Western blot or immunofluorescence.</p> Results <p>SSD dose‑dependently improved liver function and reduced hepatocyte necrosis, collagen deposition, and apoptosis. It attenuated oxidative stress injury, inflammatory responses, and endoplasmic reticulum stress (ERS) in BDL mice. SSD also mitigated bile acid‑induced injury and ERS in HepG2 cells. Moreover, SSD suppressed the activation of the TLR4/MyD88/NF‑κB/NLRP3 pathway. The TLR4 inhibitor TAK‑242, similar to SSD, ameliorated bile acid‑induced HepG2 cell injury and ERS, whereas the TLR4 agonist LPS counteracted the protective effects of SSD and its inhibition of ERS in bile acid‑stimulated HepG2 cells.</p> Conclusion <p>SSD attenuates CLI, an effect that may be associated with the suppression of the TLR4/MyD88/NF‑κB/NLRP3 pathway and the subsequent inhibition of ERS.</p>