CpG-ODN stimulates dendritic cells (DCs) to produce cytokines, which are important for pathogenesis of autoimmune disorders and vaccine strategy for cancer. CpG-ODN activates the TLR9/MyD88/TRAF6 cascade leading to activation of IKK-NF-κB and JNK, which are critical for production of pro-inflammatory cytokines. However, whether other molecules are involved in activation of CpG-ODN signaling is still not clear. Here we report that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is involved in this activation process. DNA-PKcs-deficient DCs exhibited a defect in the IL-6 and IL-12 response to CpG-ODN in a dose- and time-dependent manner. Loss of DNA-PKcs impaired phosphorylation of IKK, IκBα, NF-κB and JNK in response to CpG-ODN. Interestingly, CpG-ODN was able to bind DNA-PKcs and induce its association and co-localization with TRAF6 in the absence of TLR9. Our data suggest that DNA-PKcs is a player in CpG-ODN signaling and may explain why DNA-PKcs is implicated in the pathogenic process of autoimmune disease.