Objective To study the mechanism of wortmannin (WM) in enhancing radiosensitivity of malignant glioma cells.Methods Human glioma cells (U251) were pretreated with 10 μmol/L WM for 2 h and then irradiated with 10 Gy X-rays.The changes of cell cycle and apoptosis ratio were examined.Western Blot and immunofluorescence staining were used to detect the expression and function of ATM and its target genes as well as DNA-PKcs.Results The ratio of apoptotic cells in the WM combined with X-ray treatment group (WM + IR) was increased from (5.3 ± 0.66) ％ of control group and (11.5 ± 2.0) ％ of IR group to (21.8 ± 2.4) ％ significantly (F =57.38,P ＜ 0.05).Along with the increase of apoptosis,the expression of cleave-caspase-3 was significantly increased in IR or WM + IR group compared to control group (q =12.49,19.19,P ＜ 0.05),and its enhancement in WM + IR group was more significant than that in IR group (q =6.70,P ＜ 0.05).Compared to control group,the cell cycle assay showed that the amount of U251 cells in G2/M phase was increased in IR group (q =9.67,P ＜ 0.05),and further increased in WM + IR group (q =21.25,P ＜ 0.05),indicating an obvious G2/M phase arrest.Moreover,WM could effectively inhibit the phosphorylation of ATM kinase and the activity of ATM downstream target genes of p53 and SMC1.WM also suppressed the IR-induced expressions of DNAPKcs and phospho-γH2AX (Ser139).Conclusions WM effectively increased the radiation sensitivity of malignant glioma cells through down-regulating the activity of ATM kinase and DNA-PK.