Objective To investigate the relationship between DNA damage which was considered enhancing apoptosis and metabolites after radiotheraphy in U87 cells, and explore the possible molecular mechanism. Methods Glioma cell line U87 were randomly separated into 0Gy, 1Gy, 5Gy, 10Gy and 15Gy groups, the cells were cultured 24h after exposed to X-ray, respectively. The DNA damage parameters (tail length, %DNA in tail and tail moment) were obtained in comet assay by fluorescence microscopy. Cell apoptosis rates and cell cycle distributions were analyzed by flow cytometry. Cell colony-forming rates were observed and counted under inverted phase-contrast microscopy. 1H NMR spectroscopy performed with a Bruker Avance 600 MHz spectrometer was used to determine the changes of metabolites. Results Along with the X-ray irradiation increasing, the DNA damage parameters (tail length, %DNA in tail and tail moment) demonstrated positive dose-dependent manner (P<0.05), the G1 stage of the cell cycle increased obviously(P<0.01), the colonyforming rates declined(P<0.01) and the apoptotic cells increased(P<0.01), the ratio of Lac/Cr decreased, significant changes had occurred in all doses of the cell lines(P<0.01), which linearly dependent on DNA damage (tail length, R2 = 0.910; %DNA in tail, R2 = 0.968; tail moment, R2 = 0.850). Conclusion DNA damage was aggravated with the increased exposure to X-ray irradiation, meanwhile 1H NMR spectroscopy can detect alterations of metabolites of the cells before cell apoptosis. 1H NMR spectroscopy has the potential to provide a noninvasive biomarker to predict apoptosis rates of glioma cells.