Objective: To investigate the expression of metastasis suppressor 1 (MTSS1) in cholangiocarcinoma tissues and its clinical significance. Methods: Eighty-nine patients with cholangiocarcinoma admitted from January 2012 to January 2014 were prospectively enrolled as study subjects, and meanwhile, 89 cases undergoing cholecystectomy due to cholecystolithiasis were collected as a control group. The difference in MTSS1 expression in the pathological tissues between the two groups of patients was compared, and the relations of MTSS1 expression with the clinicopathologic variables and prognosis of the patients with cholangiocarcinoma were analyzed. Results: Compared with control group, the positive rate of MTSS1 expression in study group was significantly decreased (20.22% vs. 58.43%, P=0.000), and the percentage of MTSS1 positive cells was also significantly reduced (9.47% vs. 43.95%, P=0.000). In patients with MTSS1 negative cholangiocarcinoma compared with those with MTSS1 positive cholangiocarcinoma, the tumor size was significantly increased (3.84 cm vs. 2.46 cm, P=0.000), the proportion of cases with clinical stage III or IV was significantly increased (70.42% vs. 27.78%, P=0.001), lymph node metastasis rate was significantly increased (77.46% vs. 44.44%, P=0.028), the 2-year mortality rate was significantly increased (42.25% vs. 16.67%, P=0.045) and the number of cases with low differentiated tumor cells was significantly increased (P=0.001). Logistic regression analysis showed that the loss of MTSS1 expression was an independent risk factor for death of patients with cholangiocarcinoma (P=0.000), and Wilcoxon test showed that the survival rate in patients with positive MTSS1 expression was significantly higher than that in patients with negative MTSS1 expression (P=0.041). Conclusion: The decrease or loss of MTSS1 expression is closely related to the occurrence and development of cholangiocarcinoma.