Objective: To investigate the role of Toll-like receptor 4 (TLR4) in the repairing process of bile duct injury. Methods: The TLR4 expressions in 12 specimens of bile duct tissue with biliary stricture and 4 specimens of normal bile duct tissue were determined by immunohistochemical staining. Biliary injury and repair model was established in TLR4 deficient (TLR4–/–) and wild-type (TLR4+/+) mice using clamping method, with their corresponding sham-operated mice as control, and the pathological changes and liver function status were observed in each group of mice 48 h after operation. Results: TLR4 was expressed mainly in the endothelial cells of the bile ducts, and its positive expression rate in bile duct wall with benign biliary stricture was significantly higher than that in bile duct wall of normal tissue (83.33% vs. 25.00%, P<0.01). No pathological changes were noted in the liver and bile duct tissues in the two groups of sham-operated mice, while obvious liver and bile duct injuries were seen in model groups of both TLR4–/– and TLR4+/+ mice, but the injuries were markedly milder in the former than those in the latter; compared with corresponding sham-operated control, the serum levels of alanine transaminase, and total or direct bilirubin in model groups of both TLR4–/– and TLR4+/+ mice were significantly increased (all P<0.05), but the increasing amplitudes of these parameters in the former were significantly slighter than those in the latter (all P<0.05). Conclusion: TLR4 contributes critically to benign bile duct stricture probably via participating in the natural immune responses of biliary epithelial cells, initiating the expressions of a range of inflammatory cytokines, and prompting fibroblast proliferation.