Abstract: Objecitve To designed antisense oligodeoxynucleotides (AS-ODNs) to reduce the expression of ATM and to study its effect on the apoptosis of Hep-2 (human epidermoid laryngeal carcinoma) cells treated with radiation in vitro. Methods Experiment was divided into AS-Lipo, Sen-Lipo, Mis-Lipo, Lipo and Hep-2 group.The expression of ATM mRNA in Hep-2 cells was examined by real-time quantitative PCR. About 18 hours after transfection, they were irradiated simultaneously with different doses of X-ray radiation (0, 2, 4, 6, and 8 Gy) respectively. Clonogenic survival assay was carried out to detect the survival ability of Hep-2 cells after irradiation. After exposed to 4 Gy radiation, flow cytometry was carried out to analyze the cell apoptosis. Results The relative ATM mRNA expression in Hep-2 cells treated with ATM AS-ODNs was decreased to (11.03±2.51)% which was much lower than that of untreated cells (P<0.05). After irradiation, the survival fraction (SF) of cells treated with ATM AS-ODNs was lower than that of other groups at the same dose of radiation. There was statistical significance between the group treated with ATM AS-ODNs and other groups (P<0.05). The apoptotic rate for the group irradiated with ATM AS-ODNs was (30.7±1.31)%, which was significantly higher than that of others (P<0.05). Conclusion AS-ODNs of ATM reduce ATM mRNA expression and enhance Hep-2 cells apoptosis to radiation in vitro.