Objective: To study the radiosensitization effects of pemetrexed disodium and gefitinib on human lung adenocarcinoma cells. Methods: Human lung adenocarcinoma A549 cells were treated with pemetrexed and/or gefinitib, and cell proliferation was examined by MTT. Colony formation was performed to detect growth inhibition effects of pemetrexed and/or gefinitib combined with radiation on A549 cells. Flow cytometry was performed to detect the changes of cell cycle and apoptosis. Expressions of intracellular Akt and p-Akt were detected by Western blot. Results: Pemetrexed and gefitinib inhibited cell proliferation of human lung adenocarcinoma A549 cells. Compared with cells treated with radiation only, radiation+pemetrexed, and radiation+gefitinib, the Dq, D0 and SF2 of cells treated with radiation+pemetrexed+gefitinib were significantly decreased. The SERs of cells treated with pemetrexed, gefitinib, and pemetrexed+gefitinib were 1.17, 1.48 and 2.62, respectively. Both pemetrexed and gefitinib induced G1/G0 phase arrest. When combined with radiation, the proportion of S phase cells was further reduced. Both pemetrexed and gefitinib enhanced radiation-induced apoptosis by decreasing p-Akt level. The p-Akt level was lowest in the cells treated with radiation+pemetrexed+gefitinib. Conclusion: Both pemetrexed and gefitinib have radiosensitization effects on A549 cells. Combination of the two drugs significantly improves the inhibitory effect of radiation through reducing S phase cells, enhances radiation induced apoptosis, and inhibits PI3K/Akt signaling pathway. This combination regimen may provide a promising strategy for lung cancer therapy.