Cholangiocarcinoma (CCA) is a malignancy of bile ducts with a high incidence of invasion and metastasis.This disease is often detected in advanced stages because of the difficulties of early diagnosis, leading to a highmortality rate. However, biomarkers for early CCA detection are still lacking. In this study, to identify potentialbiomarker proteins, differential secretome analysis by the GeLC-MS/MS approach was applied with four CCAcell lines and a control immortalized cholangiocyte cell line. Among 78 up-regulated proteins, 53 including ICAM-1 were exclusively expressed in four CCA secretomes but not in MMNK1. Based on this result, we measuredICAM-1 levels in serum samples of CCA patients and healthy controls and found significantly higher values inCCA patients’ sera. Receiver operating characteristic curve analysis suggested that serum ICAM-1 level couldbe a discriminatory diagnostic marker for CCA and healthy controls (area under curve=0.829) with a sensitivityof 77% and a specificity of 70% at a cut off value of 167 ng/ml. Moreover, the serum ICAM-1 showed positivecorrelations with alkaline phosphatase and carcinoembryonic antigen levels. Comparison of ICAM-1 levels ofpaired pre- and post-operative sera of 12 cases revealed significant decrease after tumor resection. However,serum ICAM-1 levels were not significantly different between CCA and benign biliary diseases with mainlyinflammatory features.