Differentiating malignant from nonmalignant biliary stenoses is challenging. This could be facilitated by the measurement of cancer biomarkers in bile. We aimed at (i) identifying new cancer biomarkers by comparative proteomic analysis of bile collected from patients with a malignant or benign biliary stenosis (exploratory phase) and (ii) verifying the accuracy of the newly identified potential biomarkers for discriminating malignant versus nonmalignant biliary stenoses in a larger group of patients (confirmation phase). Overall, 66 proteins were found overexpressed (ratio>1.5) in at least one cancer condition using proteomic analysis and 7 proteins were increased in all malignant/nonmalignant disease comparisons. Preliminary screening by immunoblot highlighted carcinoembryonic cell adhesion molecule 6 (CEAM6), a cell surface protein overexpressed in many human cancers, as an interesting candidate biomarker. ELISA subsequently confirmed CEAM6 as a potential bile biomarker for distinguishing malignant from benign biliary stenoses with a receiver operating characteristic (ROC) area under the curve (AUC) of 0.92 (specificity 83%, sensitivity 93%, positive predictive value 93%, and negative predictive value 83%). No significant difference in serum CEAM6 level was found between malignant and nonmalignant samples. Combining bile CEAM6 and serum CA19-9 in a panel further improved diagnostic accuracy for malignant stenoses (AUC 0.96, specificity 83%, sensitivity 97%, positive predictive value 93%, and negative predictive value 91%). CEAM6 measurement in bile could be clinically useful to discriminate between malignant and nonmalignant causes of biliary stenosis. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.