Objective: To investigate the PROX-1 and Ki-67 expressions in cholangiocarcinoma tissues and their significance. Methods: The PROX-1 and Ki-67 expressions in cancer tissues form 46 patients with hilar cholangiocarcinoma (29 cases with lymph node metastases, and 17 cases without lymph node metastasis) and bile duct tissues from 23 cases with benign biliary disease were determined by immunohistochemical staining. The relations of PROX-1 and Ki-67 expressions with lymph node metastasis and other clinicopathologic factors of cholangiocarcinoma were analyzed. Results: Positive expression rates of PROX-1 and Ki-67 were both increased in an ascending order in bile duct tissue of benign biliary disease, cholangiocarcinoma tissue without lymph node metastasis and cholangiocarcinoma tissue with lymph node metastases, and all differences had statistical significance (all P<0.05). The positive expression rates of PROX-1 and Ki-67 were both associated with the degree of differentiation of the tumor (both P<0.05), and were not related with sex and age of the patients (all P>0.05). There was a positive correlation between PROX-1 and Ki-67 expression in cholangiocarcinoma tissue (r=0.831, P<0.05). Conclusion: PROX-1 and Ki-67 over-expressions are closely related to the malignant biological behavior of cholangiocarcinoma, and furthermore, there is relevance between their expression levels.