Background: Tumor radioresistance leads to a reduction in the efficiency of radiation therapy. It is very important to explore the cellular mechanisms leading to radioresistance and to find potential therapeutic targets, which might improve the efficacy of radiation therapy. This study was to investigate the role of ataxia-telangiectasia mutated (ATM) and murine double minute X (MDMX) in radioresistance in non-small cell lung cancer A549 cells and their corresponding mechanisms of action. Materials and Methods: Non-small cell lung cancer A549 cells were irradiated with X-rays in the presence or absence of ATM inhibitor. Cell survival, cell apoptosis, cell proliferation, mRNA of ATM and MDMX, and protein expression of ATM, MDMX, γ-H2AX, Caspase3, and Beclin1 were measured. Results: After the inhibitor (KU60019) treatment combined with X irradiation, the A549 cells showed a significant decrease in colony formations compared to the group received irradiation alone. The MDMX knockdown A549 cells showed a significant increase in colony formations compared to the control group. ATM downregulated the expression of MDMX after irradiation treatment in A549 cells. Irradiation led to a significant increase in γ-H2AX expression, but MDMX knockdown decreased the γ-H2AX expression after irradiation. The change of Caspase3 expression was the same as γ-H2AX. Irradiation led to a significant increase of Beclin1 expression and MDMX knockdown increased the Beclin1 expression after irradiation. Conclusion: This study indicated that ATM induced radioresistance through downregulating the expression of MDMX, which was at least partly associated with the activation of autophagy and the decrease of DNA damage in A549 cells.