Unmethylated cytosine-phosphorothioate-guanine (CpG)-containing oligodeoxynucleotides (ODNs) are synthetic DNA sequences that mimic bacterial DNA, and are known to serve as ligands for Toll-like receptor 9 (TLR9). The interaction between a CpG ODNs with TLR9 activates the complex downstream cascade that contributes to exerting its function. In the present study, the results of clonogenic assays demonstrated that the activation of TLR9 by CpG ODNs significantly increased the radiosensitivity of A549 lung cancer cells, with a sensitivity enhancement ratio (SER) of 1.28. When the expression of TLR9 was effectively silenced, CpG ODNs used alone were identified to produce SERs as low as 1.01. Flow cytometry demonstrated that the interaction between TLR9 and CpG ODN 7909 alone did not significantly affect the rate of apoptosis, but may significantly enhance the radiation-induced apoptosis of A549 cells. Western blot analysis revealed that TLR9 activation by CpG ODN 7909 increased the levels of mitogen-activated protein kinase 14, cellular tumor antigen p53, B-cell lymphoma 2 associated X protein and genome polyprotein, and decreased Bcl-2 expression levels, whereas these effects were not observed in CpG ODN 7909-treated cells in which TLR9 was knocked down. These results suggest that CpG ODN 7909 may enhance radiosensitivity through TLR9 activation, and partially via the p53 pathway in A549 lung cancer cells.