Intrahepatic cholangiocarcinoma (ICC), a malignant tumor derived from the bile duct epithelium, is one of the leading causes of death from cancer, worldwide. However, the mechanisms related to it remain largely unknown. In this study, an analysis of the gene expression profiles for ICC was done using the frequency of the ESTs obtained from nine cDNA libraries that constructed from 4 ICC cell lines and 4 normal liver tissues. One hundred and thirty-seven genes were identified as being either up- or down-regulated in human ICC cells. Thirty genes were randomly selected to confirm their differential expression in 4 human ICC cell lines and 5 ICC tissues compared to normal liver tissues by semi-quantitative RT-PCR. Among these genes, ANXA1, ANXA2, AMBP, and SERPINC1 were further verified by immunohistochemical analyses. In conclusion, these identified genes represent potential biomarkers for ICC and represent potential targets for elucidating the molecular mechanisms that are associated with ICC.