Background/Aims: MicroRNAs are a small non-coding family of genes involved in the regulation of gene expression in a post-transcriptional manner and contribute to cell proliferation, differentiation and apoptosis. Our aims were to identify statistically unique miRNA profiles in human intrahepatic cholangiocarcinoma for diagnosis and investigate their specific involvement in various cell biological processes in cholangiocarcinoma. Methods: Laser capture microdissection techniques and TaqMan miRNA assays for mature miRNAs were performed to assess the genomewide expression of miRNAs in 27 human ICCs, 10 normal cholangiocyte cells and 8 normal liver tissues precisely and quantitatively. Two selected miRNAs, mir-204 and mir-320, were introduced into cholangiocarcinoma cell lines to examine their effects on potential target genes, Bcl-2 and Mcl-1, respectively. Results: A cluster of 38 miRNAs was markedly distinguishable between tumor and normal tissues. At least two distinct clusters of tumor samples could be identified that were associated with the higher or lower expression levels of carbohydrate antigen 19-9. Moreover, the exogenous expression of mir-320 or mir-204 could negatively regulate Mcl-1 or Bcl-2 expression and facilitate chemotherapeutic drug-triggered apoptosis. Conclusions: miRNA expression profiles are closely associated with the biological and clinical behavior of ICC. The modulation of aberrantly expressed miRNAs might prove a promising therapeutic strategy. © 2008 European Association for the Study of the Liver.