Metformin, one of the most widely used antidiabetic drugs, has recently been associated with potential antitumorigenic effects. In this study, we evaluated the possible cytotoxic impact of combined low doses of metformin and ionizing radiation (IR) on 2 human hepatoma cell lines. The cytotoxic effect of metformin combined with IR was subsequently determined by clonogenic survival and cell cycle assays, assessment of mitochondrial complex I and lactate dehydrogenase (LDH) activity, measurement of cellular adenosine triphosphate (ATP) levels, comet assay and analyses of the formation and disappearance of phosphorylated histone H2AX (γ-H2AX) protein. The combination of metformin and IR caused a much stronger cytotoxicity than the treatment with metformin or IR alone, leading to an ~80% decrease in cell viability and ~35% increase in the accumulation of cells in the G2/M phase of the cell cycle in the 2 hepatoma cell lines. In addition, a reduction in mitochondrial complex I activity (~70%) and a significant increase in LDH activity, as well as lactate production were observed in the cells exposed to metformin. Interestingly, a severe depletion in ATP, increased olive tail moment and the delayed disappearance of γ-H2AX expression were detected in the hepatoma cells treated by metformin plus IR. These findings show that the combination of a low concentration of metformin and IR results in the considerable enhancement of cytotoxic effects in human hepatoma cell lines, leading to decreased DNA repair by reducing ATP production. The data provided in this study may elucidate the remarkable efficiency of this combination treatment and suggest that metformin may be used as a potential adjunct to the radiotherapy of hepatocellular carcinoma.