Objective To investigate the role of KiSS-1 gene in the metastatic process of carcinoma of gallbladder and the clinicopathologic significance of KiSS-1 gene expression in carcinoma of gallbladder. Methods Pathological specimens from 59 gallbladder carcinoma tissues (13 hepatic invasion and 13 lymphatic invasion tissues were included), matched with 7 para-tumor and 6 normal gallbladder tissues, were examined for the expression of KiSS-1 gene by tissue microarray technique and immunohistochemistry (EnVision). Results The positive rate of KiSS-1 expression was down-regulated (P＜0.05) in tumor tissues, as compared with normal and para-tumor tissues. In carcinoma of gallbladder, the expression of KiSS-1 had no relationship with the gender, age, tumor size, histological grade or differentiation, and metastasis of lymph node, while was associated with the depth of infiltration, invasion of liver and the clinical stages (Nevin). In Ⅰ+Ⅱ, Ⅲ+Ⅳ and Ⅴ stage, the positive rates of KiSS-1 were 92.3%, 57.1% and 27.8% respectively, with an undeniably clear lowering tendency (P=0.002). Conclusion Down-regulating expression of KiSS-1 is closely associated with the processes of genesis, invasion and metastasis in carcinoma of gallbladder, and may participate in regulating these processes.