Objective: To study the expression of blood vessel epicardial substance (BVES) in human gallbladder cancer tissue and its clinical significance. Methods: The BVES expressions in gallbladder tissue specimens form 65 patients with gallbladder cancer and 55 cases with benign gallbladder diseases were determined by immunohistochemical staining, and the relations of BVES expression with clinicopathological profiles of gallbladder cancer were analyzed. Results: The high expression rate and low expression rate of BVES in gallbladder cancer tissues was 20.0% (13/65) and 80.0% (52/65), which was 74.5% (41/55) and 25.5% (14/55) in benign gallbladder tissues respectively, and the difference between the two groups had statistical significance (χ2=35.811, P<0.05). The univariate analysis indicated that the BVES expression was closely associated with the degree of differentiation, clinical stage and lymph node metastasis of the gallbladder cancer, as showed by the statistical differences of these three factors between the two groups (all P<0.05). Conclusion: The low or lack of BVES expression may be closely connected to the occurrence and development of gallbladder cancer.