Methotrexate is a folic acid antagonist that has been shown to be genotoxic to normal healthy cells. Metformin is an insulin-sensitizing agent, with multiple potential pharmacodynamic profiles. The aim of the present study was to evaluate the genotoxic effect of methotrexate on DNA and the potential ameliorative effect of metformin on chromosomal damage induced by methotrexate. The present study was performed in vitro, and the frequency of chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) in human cultured lymphocytes were measured. Blood samples from five non-smoking healthy men aged 20-35 years were donated and used in the present study. Treatment of cultured blood cells with methotrexate significantly increased the number of cells with CAs (P<0.0001) and the frequency of SCEs (P<0.0001). The chromosomal injury induced by methotrexate was significantly reduced by pretreatment of the samples with metformin (P<0.0001). Importantly, the treatment of the cells with metformin alone did not affect the frequency of SCEs compared with the control group (P>0.05). Additionally, methotrexate and metformin alone, and combined, induced significant decreases in the proliferative index compared with the control group (P<0.05). In conclusion, metformin ameliorated the genotoxicity induced by methotrexate in cultured human lymphocytes.