Background: Distinguishing bile duct carcinoma from other diagnoses is often difficult using endoscopic or percutaneous techniques. The cell surface protein CEACAM6 is over-expressed in many gastrointestinal cancers and may be selectively elevated in biliary adenocarcinoma. The aim of the present study is to determine if CEACAM6 can be detected in the bile of patients with biliary cancer and can serve as a diagnostic biomarker for cholangiocarcinoma. Methods: Bile duct and gallbladder bile from patients with benign biliary disease and cholangiocarcinoma (hilar, intrahepatic and distal) was collected at the time of index operation. The concentration of CEACAM6 was quantified by sandwich enzyme-linked immunosorbent assay (ELISA) and correlated to pathologic diagnosis. Diagnostic capability of CEACAM6 was evaluated by Wilcoxon rank-sum, logistic regression, and receiver operating characterisitic (ROC) curve analysis. Results: Bile from 73 patients was analyzed: 40 with benign disease and 33 with cholangiocarcinoma. Patients in the benign cohort were younger, predominantly female, and had lower mean biliary CEACAM6 levels than patients in the malignant cohort (102 ng/ml vs. 239 ng/ml; p=0.0006). Logistic regression analysis determined CEACAM6 to be a positive predictor of extrahepatic but not intrahepatic cholangiocarcinoma (p=0.0015). ROC curve with a CEACAM6 level >22.1 ng/ml was associated with 85% sensitivity, 73% specificity, and a likelihood ratio of 3.15 (AUC 0.82) for extrahepatic cholangiocarcinoma. Conclusions: CEACAM6 levels in bile can predict patients with extrahepatic cholangiocarcinoma with acceptable accuracy. Further investigation is warranted in a larger cohort of patients prior to clinical application.