Abstract: Objective In this study, we specifi cally investigated the effect and the mechanism of gefi tinib, the tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR), on radiosensitizing the nonsmall cell lung cancer (NSCLC) A549 and H1975 cell lines. Methods Two cell lines of NSCLC A549 and H1975 were divided into two groups, the X ray and X-ray plus gefi tinib groups. The former was irradiated with X rays only, and the latter was treated with 10μmol/L gefitinib for 24 h before irradiation under the same conditions. The cells were tested by cloning formation assay to identify the radiosensitivity of both groups. Immunostaining for confocal microscopy was used to observe the localization of nuclear γ-H2AX foci at different time points after irradiation. Nuclear EGFR expression was detected by Western blot after radiotherapy. Results The cloning formation assay revealed that the surviving fraction at 2 Gy (SF2) of the gefi tinib-interfering group (0.3475) was lower than that of the X ray group (0.4833) in A549 cells. There was no signifi cant difference between the SF2 values of the two respective groups in H1975 cells (0.3094 vs 0.3207). The confocal microscopy results found that the average number of γ-H2AX foci in the X ray plus gefi tinib group was more than that in the X ray group on every time point after 4 Gy irradiation in A549 cells. But there was no remarkable difference of the average number of γ-H2AX foci in H1975 cells between the two groups. Based on Western blot analyses, EFGR protein of A549 cells translocated into the nuclear in the X ray group after 4 Gy irradiation, but most of those located in cytoplasm in the X ray plus gefi tinib group. However, EGFR protein expressed in the nuclear of H1975 cells, seldom in the nucleus, for both treatment groups. Conclusion Gefi tinib enhances the radiosensitivity of A549 cells, which may be attributed to the suppression of EGFR transport into the nucleus to recover the double strand break(DSB). However, gefi tinib does not affect H1975 cells, related to EGFR remains in the cytoplasm after irradiation.