Abstract: As a widely used carbamate insecticide, the toxic effects of methomyl on the environmental non-target organisms have been paid more attention. The genotoxicity of methomyl against Drosophila Schneider 2 (S2) cells and Human hepatocellular carcinoma G2 (HepG2) cells was investigated by using MTT (3-4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay procedure, single-cell comet electrophoresis and γH2AX (phosphorylated histones H2AX) immunofluorescence. The results showed that as the concentrations of methomyl were increased, cell viability of S2 and HepG2 were gradually declined; cell comet phenomena were induced significantly, the length of comet tail became longer and the tail area increased; the percentage of γH2AX positive cells were increased gradually. The results suggested that the extent of DNA damage in S2 and HepG2 cells was concentration-dependent on the doses of methomyl, and methomyl could induce DNA double-strand breaks and cause cell apoptosis. Methomyl possessed potentially genotoxicity on insect and human, the prolonged exposure to methomyl might be harmful for human health.