Objective: To investigate the influence of TNF-α on activity of P311/TGF-β1/α-SMA signaling pathway in rabbit bile duct fibroblasts and the interventional effect of tetramethylpyrazine (TMP). Methods: Bile duct fibroblasts of rabbits were isolated and cultured, and then identified. The bile duct fibroblasts were exposed to TNF-α or TNF-α plus different concentrations (0.08, 0.4 and 2.0 mg/mL) of TMP respectively for 48 h, using untreated bile duct fibroblasts as blank control. Afterwards, the cell proliferation was determined by CCK-8 assay, mRNA expressions of P311, TGF-β1 and α-SMA were measured by real-time PCR, and protein expressions of TGF-β1 and α-SMA were examined by Western blot analysis. Results: In bile duct fibroblasts after TNF-α treatment, the cell proliferation significantly accelerated, and the mRNA expressions of P311, TGF-β1 and α-SMA as well as the protein expressions of TGF-β1 and α-SMA were significantly up-regulated compared with blank control cells (all P<0.05); TMP inhibited the above effects of TNF-α with a concentration-dependent tendency, and the inhibitions exerted by TMP at 0.4 and 2.0 mg/mL were significant (all P<0.05). Conclusion: TNF-α can promote proliferation of bile duct fibroblasts possibly via regulating P311/TGF-β1/α-SMA signaling pathway in fibroblasts, and TMP can inhibit the activation of this pathway induced by TNF-α, so it may have preventive and therapeutic effect on benign biliary stricture.