Abstract: To investigate the efficacy of h-R3 combined radiotherapy with different treatment schedules on human lung cancer cells expressing different EGFR levels. Methods: The in vitro cultured human lung cancer cell lines A549 and Calu-6 were used as research objects. Western blot assay was conducted to determine the expression of EGFR in the cell lines. The grouping of h-R3 administration was as follows: Group I, administration 24 h before irradiation; Group II, administration during irradiation; and Group III, administration 24 h after irradiation. The cytotoxic and radiosensitizing effects of h-R3 on the two cell lines were determined by MTT reduction and clone formation assay. The cell apoptosis and cell-cycle distribution were detected using flow cytometer. The expression of P21, Bax, Bcl-2, phosphorylated DNA-PKcs, Akt, and pAkt were determined by Western blot analysis. Results: The IC20 and IC50 values were ( 1.13±0.37 ) and ( 24.78±1.25 ) μg/mL, respectively, 24 h after h-R3 administration in the A549 cells. Moreover, the radiosensitization ratios of the administration before, during, and after the radiation therapy were 2.02, 1.77 and 1.39, respectively. Higher apoptotic rate and G2/M blockage were observed in the group administered before radiotherapy; in addition, the changes in the expression of P21, Bax, Bcl-2, phospho-DNA-PK, and pAkt were more obvious in this group than in the others. The IC20 and IC50 values were ( 3.42±1.26 ) and ( 51.15±2.41 ) μg/mL, respectively, 24 h after h-R3 treatment in the Calu-6 cells. The radiosensitization ratios of the administration before, during, and after the radiation therapy were 1.23, 0.99 and 0.91, respectively. The apoptotic rate and G2/M blockage achieved the highest proportion in the group administered before the radiotherapy. In the group with h-R3 administration before radiotherapy, there were no apparent changes in the expressions of P21, Bax, Bcl-2, phospho-DNA-PK, and pAkt compared with the other groups. Conclusion: The best radiosensitizing effect was achieved when h-R3 was delivered before irradiation. The h-R3 differently enhanced the radiosensitizing effects in the two cells, probably via Akt/or pAkt expression. The inhibitory action of h-R3 on cell proliferation occurred both in the A549 and in the Calu-6 cells. The radiosensitizing effect was affected by different time series of administration, in which the effect of the administration before radiotherapy ranked the best.