Intrahepatic cholangiocarcinoma (ICC), which arises in the small bile ducts of the liver, is the second most common liver malignancy. Although modulation of microRNA (miRNA) expression has been shown to be a potent sign of malignant tumors, miRNA profiles of ICC remains unclear. We performed sequencing analysis of the small RNA libraries of 2 ICC cell lines (HuCCT1 and MEC) and one normal intrahepatic biliary epithelial cell line (HIBEpiC) to produce the miRNA profiles of ICC in vitro. Furthermore, by means of the real-time polymerase chain reaction (PCR) we validated the differential expression of miRNAs cloned exclusively or predominantly from each of the cell lines. A total of 35,759 small RNA clones were obtained from the 3 cell lines. We identified 27 miRNAs that were expressed exclusively or predominantly in each cell line. Subsequent validation with the real-time PCR confirmed that the miRNAs hsa-miR-22, -125a, -127, -199a, -199a*, -214, -376a, and -424 were expressed specifically in HIBEpiC but were downregulated in the ICC cell lines. Our study provides important information for facilitating studies of the functional role(s) of miRNAs in carcinogenesis of the hepatobiliary system. The biliary epithelial cell-specific miRNAs identified in this study may serve as potential biomarkers for ICC.