Background: Inducible nitric oxide synthase (iNOS) has supposed to implicate in inflammation, infection, liver cirrhosis, and neoplastic diseases. This study was designed to explore the biological and clinical function of iNOS in intrahepatic cholangiocarcinoma (ICC). Methods: RT-PCR (Real-time quantitative PCR) and immunohistochemical staining were used to analyze the expression of iNOS in ICC and adjacent tissues. CCK8, transwell assays, flow cytometry were conducted to detect the proliferation, apoptosis, cell cycle. Western blotting was performed to detect the expression of target proteins. Multivariate analyses were conducted to analysis associates between clinicopathological values and survival. Results: We found that levels of iNOS mRNA and protein were dramatically increased in ICC samples and positively correlated with complicated bile duct stone, differentiation, pathology T, pathology M, Wip1, MMP-2, and MMP-9. iNOS expression was significantly correlated with the poor survival of ICC patients. Furthermore, iNOS was high expression in ICC cell lines (QBC-939, ICC-9810, SSP-25) compare with human normal biliary epithelium cell line (HIBEpic); both iNOS knockdown and iNOS inhibitor (1400 W) suppressed cell proliferation, invasion, and migration though nitric oxide production in ICC cells. Down-regulation of iNOS also induced G0/G1 cell cycle arrest and ICC cell apoptosis. Moreover, iNOS knockdown treatment significantly decreased Wip1, MMP-9, and MMP-2 gene expression. Conclusion: Lowly expressed iNOS-inhibited proliferation yet promoted apoptosis of ICC cells. Our data show targeted inhibition of iNOS in ICC may have therapeutic value.