Objective: To investigate the effect of shRNA-mediated nerve growth factor β (NGF-β) down-regulation on proliferation and apoptosis of the human cholangiocarcinoma cell line QBC939 in vitro. Methods: The stable QBC939 cells transfected with the NGF-β shRNA were constructed. Then, the QBC939 cells were divided into interference group (transfected with NGF-β shRNA) and control group (transfected with empty plasmid). The effect of transfection was determined by Western blot method. The proliferative and colony formation ability of the QBC939 cells was assessed by CCK-8 assay and sphere formation assay, respectively. The cell cycle and apoptosis of the QBC939 cells were detected by flow cytometry. Results: Compared with the control group, the QBC939 cells of interference group presented markedly deceased ability of cell proliferation and colony formation, and significantly reduced ratio of S-phase cells (P<0.05) and increased apoptotic rate (P<0.05). Conclusion: NGF-β inhibition can decrease the proliferative and colony formation ability of QBC939 cells, which is related to the action of S-phase block and apoptosis enhancement.