The purpose of present study was to investigate the radiosensitivity of lung cancer cells affected by human lung cancer-associated fibroblasts (CAFs) in transwell and direct co-culture in vitro. Human lung CAFs was obtained from fresh human lung adenocarcinoma tissue specimens by primary culture with tissue explants method, and was identified by immunofluorescence staining. The radiosensitivity of A549 and H1299 lung cancer cells in mono-culture, transwell and direct co-culture with human lung CAFs were investigated by clonogenic survival assay, respectively. Human lung CAFs were obtained successfully, and had specific immuno-staining with α-SMA, Vimentin, and FAP, and no cytokeratin-18 expression. The plating efficiency (PE) of A549 cells without receiving irradiation were 0.206±0.031, 0.210±0.007 (>0.05) and 0.352±0.023 (<0.05) in mono-culture, transwell and direct co-culture, respectively. The PE of H1299 cells were 0.200±0.039, 0.259±0.024 (>0.05) and 0.323±0.025 (<0.05) in mono-culture, transwell and direct co-culture, respectively. The clonogenic survival assay showed that the protection enhancement ratio of human lung CAFs in transwell and direct co-culture were 1.11 and 1.29 for A549 cells, and 1.15 and 1.25 for H1299 cells, respectively. The results suggested that human lung CAFs promote the radioresistance of lung cancer cells significantly. Its radioprotective effect may attribute to CAFs stimulating lung cancer cell proliferation.