Objective: To investigate the effect of RNA-mediated interference of inducible nitric oxide synthase (iNOS) gene expression on growth of human cholangiocarcinoma cells. Methods: Three iNOS-siRNA sequences targeting iNOS gene (siRNA1, siRNA2 and siRNA3) and a negative control siRNA sequence were designed and synthesized, and were respectively transfected into human cholangiocarcinoma QBC939 cells, and then, the transfection efficiencies were determined by fluorescence microscopic observation, and the interference effects were examined through changes in iNOS mRNA and protein expressions. The iNOS-siRNA sequence with most evident interference efficacy was selected for use to observe the changes in proliferation, cell cycle and apoptosis in QBC939 cells after its transfection. The untreated QBC939 cells served as blank control. Results: All the three synthesized iNOS-siRNA sequences were effectively transfected into QBC939 cells, and iNOS mRNA and protein expression levels were all significantly reduced in QBC939 cells after their transfection (all P<0.05), in which, siRNA2 showed the most evident inhibitory effect on iNOS. The negative control siRNA sequence exerted no significant effect on iNOS mRNA and protein expressions in QBC939 cells (both P>0.05). In QBC939 cells after siRNA2 transfection, the proliferation was significantly decreased, with significant G0/G1 phase arrest and increased apoptosis rate (all P<0.05), while no significant changes were noted in above indexes in QBC939 cells after transfection of negative control siRNA sequence (all P>0.05). Conclusion: RNA interference can effectively decrease iNOS gene expression in cholangiocarcinoma cells, which thereby inhibit the proliferation and promote apoptosis of cholangiocarcinoma cells.