Stenosis of the common bile duct may be either due to benign (chronic pancreatitis) or malignant (cholangiocarcinoma, pancreatic adenocarcinoma) conditions. The benign nature of the stricture should be first confirmed in order to ensure appropriate therapy. Therefore, the identification of markers allowing discrimination between malignant and benign biliary stenosis would be very valuable in clinical practice. To this intent, we performed a proteomic analysis of bile samples from patients having a biliary stenosis caused by pancreatic adenocarcinoma. Bile samples were collected during endoscopic retrograde cholangiopancreatography and purified using different methods. The extracted proteins were then analyzed by SDS-PAGE and LC-MS/MS. A total of 127 proteins were identified, 34 of which have not been previously detected in proteomic studies of bile. Among them, several proteins have been described as potential biomarkers of pancreatic cancer. We extended our investigation by studying the expression of some of these pancreatic cancer markers in bile samples collected from patients with various etiologies of biliary stenosis including pancreatic cancer, cholangiocarcinoma, chronic pancreatitis, as well as gallstone-induced stenosis. Our data showed a conspicuous overexpression of CEACAM6 and MUC1 (CA19-9) in pancreatic cancer and cholangiocarcinoma samples, according to the hypothesis that bile fluid collects cancer-associated protein leaking from the tumor microenvironment. These results underline the interest of using bile as a source of biomarkers for the diagnosis of malignant biliary stenosis.