Objective: To investigate the effect of metformin on cell proliferation, apoptosis and cell cycle in human cholangiocarcinoma cells and the mechanism. Methods: Human cholangiocarcinoma RBE cells were exposed to metformin, Compound C (AMPK inhibitor) or metformin plus Compound C respectively, using the untreated RBE cells as blank control. The cell proliferation was determined by MTT assay, apoptosis and cell cycle were measured by flow cytometry, and the expressions of proteins associated with AMPK/mTOR signaling pathway were detected by Western blot analysis, respectively. Results: In RBE cells treated with metformin compared with blank control, the survival rate was decreased, apoptosis rate was increased, the ratio of G0/G1 cells was increased while ratio of S cells was decreased, and the phosphorylated AMPK (p-AMPK) level was increased while the phosphorylated mTOR (p-mTOR) was decreased, and all differences had statistical significance (all P<0.05). The above effects exerted by metformin were all abolished by the combination treatment of Compound C, and the differences in all tested indexes had no statistical significance compared with blank control group (all P>0.05). All the indexes in RBE cells treated with Compound C alone had no statistical significance compared with blank control group (all P<0.05). Conclusion: Metformin can inhibit cell growth and promote apoptosis and cell cycle arrest in human cholangiocarcinoma RBE cells, and the mechanism may be associated with its activating AMPK and thereby suppressing mTOR downstream effector molecules.