Abstract: Objective To investigate the effects of ionizing radiation combined with gefitinib on the resistance mutations, apoptosis and expression of apoptosis-related proteins of non-small cell lung cancer(NSCLC) cell line H1975, which had acquired drug resistance, as well as to explore the possible mechanism. Methods The relative quantitation of T790M mutation in H1975 cells treated by different methods was analyzed by real-time PCR. Apoptosis rates in different treatment groups were detected by cytometry (Annexin V-FITC). The expressions of apoptosis-related proteins were detected by Western blot. Results The T790M mutation of H1975 cells in 2.5Gy ionizing radiation group dropped by 0.67 times, compared with 0Gy normal control group; the T790M mutation was reduced as the radiation doses was increased(P＜0.05). The rates of cell apoptosis in ionizing radiation combined with gefitinib group (44.35±8.49)% was statistically higher than those in ionizing radiation group(21.84±5.62)% and gefitinib group(17.38±6.78)% (P＜0.05). The expressions of phosphorylated epidermal growth factor receptor(p-EGFR) and phosphorylated protein kinase B(p-AKT) proteins were significantly decreased by the combination of ionizing radiation and gefitinib. Conclusion For NSCLC cell line H1975 with acquired drug-resistance, ionizing radiation could overcome gefitinib-resistance. The combination of ionizing radiation and gefitinib shows a great synergistic effect.