Abstract: Objective To study mitomycin-induced DNA damage and proliferation inhibition of gastric cancer cells by detection of γH2AX in vitro. Methods Mitomycin with different concentrations was used to incubate gastric cancer cell line SGC-7901 cells in vitro.Focus formation and protein levels of γ H2AX were detected by immunofluorescence and western-blotting techniques, respectively, MTT assay was used to detect the proliferation of gastric cancer cell. Results The mitomycin existed concentration and time interaction effect. Except 400 μg/ml, the average number of γH2AX foci and focus cell rate were gradually increased (P<0.05). At 400 μg/ml, in 6 h group average number of γH2AX foci and focus of cell rate were less than those in 4 h group (P<0.05). With time of mitomycin treatment prolonged,γH2AX protein levels were increased and then decreased (P<0.05). MMC significantly inhibited the proliferation of SGC-7901 (P<0.05). Conclusion γH2AX was a sensitive marker to predict DNA damage and proliferation inhibition of gastric cancer cell induced by Mitomycin C in vitro.