The secretin dependent biliary secretion of ions and water by transporters and/or channels, which located at apical membrane of cholangiocytes, is essential for the regulation of bile flow. The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in the chloride secretion into the bile. In the cystic fibrosis (CF) patients, totally 5 to 10% of patients develop the progressive biliary fibrosis resembling primary sclerosing cholangitis. The loss of cystic fibrosis transmembrane conductance regulator (CFTR) ion channel function leads to the onset of liver disease in human. The ezrin, radixin and moesin (ERM) proteins are identified as cross-linkers between the plasma membrane proteins and actin cytoskeleton. Ezrin interacts with NHERF1 via its N-terminal binding domain and crosslink it with actin cytoskeleton via its C-terminal binding domain. On the other hand, CFTR interacts with NHERF1 via its c-terminal PDZ binding motif. In the cholangiocyte, ezrin, but not radixin or moesin, is exclusively expressed and colocalizes with CFTR and NHERF1 at apical membrane of cholangiocyte. Here we investigated that physiological role of ezrin in the regulation of biliary chloride secretion by CFTR in vitro. In the normal immortalized cholangiocytes, ezrin colocalized with CFTR and NHERF1 at the apical membrane. The apical surface expression of CFTR was examined by immunofluorescent staining and surface biotinylation. In the normal and wild type ezrin transfected cholangiocytes, CFTR colocalized with NHERF1 and ezrin at apical surface. However, the surface expression of CFTR was significantly reduced in the dominant negative (DN) ezrin transfected cholangiocyte. The dibutyryl cAMP treatment increased the surface expression of CFTR in DN-ezrin expressing cholangiocyte whereas it was still lower than normal and wild type ezrin transfected cells, suggesting that ezrin plays an important role for the apical targeting of CFTR. Consistent with the reduction of surface expression of CFTR, chloride efflux activity was also decreased in the DN-ezrin expressing cholangiocytes. In conclusion, ezrin is required for the correct apical membrane localization of CFTR in the cholangiocyte.