Objective: To investigate the effect of inducible nitric oxide synthase (iNOS) inhibitor on the biological behaviors of cholangiocarcinoma cells. Methods: Human cholangiocarcinoma QBC939 cells were cultured in different concentrations of iNOS inhibitor 1400W for 24 h, and then, the NO contents and proliferation status of each group of cells were determined by nitrate reductase assay and MTT assay respectively, and the half-inhibitory concentration (IC50) was also calculated. After that, QBC939 cells were exposed to an appropriate concentration of 1400W, according to the IC50 value, for 24 h, and then, the cell migration and invasion were detected by wound healing assay and Transwell invasion assay, respectively. QBC939 cells cultured with the medium without 1400W were used as blank control for all experiments. Results: Compared with blank control group, the NO content and proliferation in each 1400W treatment group were significantly reduced, with a concentration-dependent trend, and the IC50 was 51.24 μmol/L. In QBC939 after exposure to 50 μmol/L 1400W, the healing rate of the scratch wound (61.7% vs. 92.3%) and number of invaded cells (72.7 vs. 128.0) were all significantly lower than those in blank control group (both P<0.05). Conclusion: iNOS inhibitor 1400W can suppress the growth, migration and invasion of cholangiocarcinoma cells, the mechanism may be associated with the changes in NO downstream signaling molecules.