The purpose of this study was to investigate the effect of 5-fluorouracil (5-FU) on the expression of TGF-β1, cyclinD1, and CDK4 in fibroblasts of miniature pig bile duct scar model cultured in vitro. The bile duct scar fibroblasts were replicated by incision and anastomosis in miniature pigs and bile duct scar fibroblasts were obtained. After cell identification, the bile duct scar fibroblasts were given different concentrations of 5-FU (0, 0.001, 0.01, and 0.1 mmol/L). After 48 h of intervention, CCK-8 method was used to detect the cell proliferation level of each group; Western blot was used to detect the expression of TGF-β1, cyclinD1, and CDK4 protein in each group of cells; and real-time polymerase chain reaction was used to detect the expression levels of TGF-β1, cyclinD1, and CDK4 mRNA in each group of cells. The results showed that after 5-FU intervention, the proliferation of bile duct scar fibroblasts was inhibited, and the expressions of TGF-β1, cyclinD1, CDK4 mRNA and protein in the cells were down-regulated (P<0.05), showing concentration-dependence. In conclusion, 5-FU may have a role in inhibiting bile duct scars. One of the mechanisms may be related to its ability to inhibit the expression of TGF-β1, cyclinD1, and CDK4 in bile duct scar fibroblasts. Copyright 2021 Zoological Society of Pakistan