Objective To investigate the expression of homeobox B7 (HOXB7) and proliferation cell nuclear antigen (Ki-67) in cholangiocarcinoma tissues and the clinical significances. Methods Immunohistochemical streptavid-in-peroxidase (SP) method was used to detect the expression of HOXB7 and Ki-67 in 70 cases of cholangiocarcinoma and 15 cases of non-tumor bile duct epithelium. The relationship between the expression of HOXB7 and Ki-67 and clinical progression and prognosis was analyzed. The correlation between the expression of HOXB7 and Ki-67 was analyzed by Spearman analysis method. Results The positive expression rate of HOXB7 and Ki-67 in cholangiocarcinoma tissue (57.1% and 71.4%) was significantly higher than that in non-tumor bile duct epithelium (20.0% and 26.7%; χ2=6.818, P=0.009; χ2=10.682, P=0.001). HOXB7 and Ki-67 expression was significantly correlated with clinical stage, tumor grade and lymph node metastasis. The positive expression rate of HOXB7 and Ki-67 was significantly higher in the stage Ⅲ+ Ⅳ group (χ2=4.073, P=0.044; χ2=4.488, P=0.034), in moderately or poorly-differentiated group than that in well-differentiated group (χ2=7.807, P=0.020; χ2=8.270, P=0.016) and in lymph node metastasis than that in no lymph node metastasis (χ2=6.076, P=0.014; χ2=4.667, P=0.031). A significant correlation was found between the expressions of HOXB7 and Ki-67 in cholangiocarcinoma (r=0.283, P=0.018). The post-operative mean survival time of patients was shortened with the increased expression levels of HOXB7 and Ki-67. Conclusion The abnormal expression of HOXB7 and Ki-67 may play an important role in the occurrence and development of cholangiocarcinoma. Key words: Cholangiocarcinoma; Homeobox B7; Proliferation cell nuclear antigen