Objective: Recent studies suggest that metformin, which is a member of the biguanide family and commonly used as an oral anti-hyperglycemic agent, may reduce cancer risk and improve prognosis of numerous types of cancer. However, the mechanisms underlying metformin’s anti-tumor effect on gastric, esophageal, colon, pancreas, livercancers remain unknown. The goal of the present study was to evaluate the effects of metformin on the proliferation of various cancers in vitro, and to study changes in the expression profile of microRNAs (miRNAs), since miRNAs have previously been associated with the anti-tumor effects of metformin in other human cancers. Design: The human varios cacer cell lines, such as esophageal cancer cell lines T.T, KYSE30 and KYSE70, hepatocellular carcinoma cell lines HLE, HLF HiH7, Alex, colon cancer cell lines Caco 2, WiDr, Colo 320, pancreas cancer cell lines PK-1, PK-7 and Panc 1 were used to study the effects of metformin on human various cancers in vitro. In addition, we used miRNA array tips to explore the differences between miRNAs in some cancer cells with and without metformin treatment. Results: Metformin inhibited the proliferation of al cancer cells in vitro. Metformin blocked the cell cycle in G0/G1 in vitro. This blockade was accompanied by a strong decrease of G1 cyclins, especially cyclin D1, as well as decreases in cyclin-dependent kinase 4 (Cdk4), Cdk6, and phosphorylated retinoblastoma protein (Rb). In addition, the expression of miRNAs was markedly altered with the treatment of metformin in vitro.Conclusion: Metformin inhibited the growth of human cancer cell lines, and this inhibition may have involved reductions in cyclin D1, Cdk4 and Cdk6.