The incidence and mortality of cholangiocarcinoma are increasing despite improvements in the diagnostic method. Since the sensitivity of brushing cytology for cholangiocarcinoma is not satisfactory, a novel diagnostic marker needs to be established. A recent report has suggested upregulation of the calcium-binding protein S100P in cholangiocarcinoma. The expression status of S100P in normal bile duct and cholangiocarcinoma tissues was assessed by immunohistochemistry. The expression levels of S100P mRNA in the brushing cytology samples during endoscopic retrograde cholangiopancreatography (ERCP) from benign biliary strictures and cholangiocarcinoma were assessed by real-time reverse transcription-polymerase chain reaction (RT-PCR). The sensitivity and specificity of each diagnostic strategy was compared. S100P was frequently expressed in the cholangiocarcinoma tissues, but not in the normal bile duct. The brushing cytology samples from the cholangiocarcinoma cases revealed higher expression levels of S100P compared with the benign biliary strictures. The relative expression level of S100P could determine the cholangiocarcinoma at higher sensitivity than classical cytology, and the combination of the S100P expression level and cytology yielded a sensitivity of 90.0%, with a specificity of 92.0%. Calcium-binding protein S100P is a novel marker of cholangiocarcinoma. Detecting the S100P expression levels in brushing cytology samples has a diagnostic value, which will be helpful for better diagnosis of cholangiocarcinoma.