Gallbladder cancer is the most common biliary tract cancer with poor prognosis and wide variation in incidence rates worldwide, being very high in some countries in Latin America and Asia. Treatment of type 2 diabetes with metformin causes a reduction in the incidence of cancer. Till date, there are no reports on the anti-tumor effects of metformin in gall bladder cancer. Therefore, this study evaluated the effects of metformin on the proliferation of human gallbladder adenocarcinoma cells in vitro and in vivo, as well as explored the microRNAs associated with the anti-tumor effects of metformin. Metformin inhibited the proliferation in gallbladder adenocarcinoma cell lines NOZ, TGBC14TKB, and TGBC24TKB, and blocked the G0 to G1 transition in the cell cycle. This was accompanied by strong reduction in the expression of G1 cyclins, especially cyclin D1 and its catalytic subunits including cyclin-dependent kinase 4, and in retinoblastoma protein phosphorylation. In addition, metformin reduced the phosphorylation of receptor tyrosine kinases, especially Tie-2, ALK, PYK, EphA4, and EphA10, as well as angiogenesis-related proteins, including RANTES, TGF-β, and TIMP-1. Moreover, metformin also markedly altered microRNA expression profile leading to an anti-tumor effect. Treatment of athymic nude mice bearing xenograft tumors with metformin inhibited tumor growth. These results suggest that metformin may be used clinically for the treatment of gallbladder adenocarcinoma.