Claudin-4 (CLDN4) is a tight junction protein that forms apical junctional complexes in epithelial andendothelial cellular sheets. Acting as a barrier and control of permeability are the general functions of tightjunction proteins contributing to tissue homeostasis, paracellular ion flux, and cell-cell contact. In this study,we immunohistochemically examined CLDN4 expression in liver fluke-associated cholangiocarcinomas (CCAs)with tissue microarrays. Regardless of the histological type and gross type of cancer, high expression of CLDN4was noted in precancerous hyperplastic/dysplastic biliary epithelia and CCA. To investigate functional roles ofCLDN4 in cancer progression, the effects of CLDN4 suppression by siRNA on cell proliferation, migration andinvasion were investigated in two CCA cell lines, KKU-M139 and KKU-M213. Suppression of CLDN4 expressiondid not alter cell proliferation but caused significant reduction of cell migration and invasion by both CCA celllines. Our results suggest that over-expressed CLDN4 may promote CCA expansion and metastasis.