Background: Cholangiocarcinoma (CCA), or bile duct cancer, is incurable with a high mortality rate dueto a lack of effective early diagnosis and treatment. Identifying cytoplasmic membrane proteins of invasiveCCA that facilitate cancer progression would contribute toward the development of novel tumor markers andeffective chemotherapy. Materials and Methods: An invasive CCA cell line (KKU-100) was stimulated usingTNF-α and then biotinylated and purified for mass spectrometry analysis. Novel proteins expressed wereselected and their mRNAs expression levels were determined by real-time RT-PCR. In addition, the expressionof ALCAM was selected for further observation by Western blot analysis, immunofluorescent imaging, andantibody neutralization assay. Results: After comparing the proteomics profile of TNF-α induced invasive withnon-treated control cells, over-expression of seven novel proteins was observed in the cytoplasmic membraneof TNF-α stimulated CCA cells. Among these, ALCAM is a novel candidate which showed significant highermRNA- and protein levels. Immunofluorescent assay also supported that ALCAM was expressed on the cellmembrane of the cancer, with increasing intensity associated with TNF-α. Conclusions: This study indicatedthat ALCAM may be a novel protein candidate expressed on cytoplasmic membranes of invasive CCA cells thatcould be used as a biomarker for development of diagnosis, prognosis, and drug or antibody-based targetedtherapies in the future.