CpG-DNA or synthetic oligodeoxynucleotides containing CpG motif (CpG-ODNs) strongly activate dendritic cells (DCs) and macrophages to produce proinflammatory cytokines including IL-6, IL-12 and TNF?. It has been suggested that CpG-DNA activates TLR9, which in turn recruits MyD88, IRAK4, IRAK1 and TRAF6, leading to activation of AP-1 and NF-?B, which are critical for immune gene expression. However, whether DNA binding proteins are involved in activation of the CpG-DNA pathway remains elusive. Here we demonstrate that Ku70 is involved in this process. Administration of CpG-ODN into Ku70-deficient mice and in vitro stimulation of bone marrow-derived DCs (BMDCs) as well as bone marrow-derived macrophages (BMDMs) from these mice resulted in defective induction of IL-6 and TNF?. Loss of Ku70 impaired activation of its downstream kinases, the I?B kinase (IKK) and the mitogen-activated protein kinases (MAPKs), by CpG-ODN. Intriguingly, in macrophages ablation of Ku70 largely abrogated IRF1 expression and subsequent IL-12 response to CpG-ODN; in BMDCs Ku70 deficiency only reduced IL-12 response to CpG-ODN at a low dose within early treatments, and had no apparent effects on this response in late treatments. Thus, our results suggest that Ku70 is important for differential regulation of inflammatory cytokine response to CpG-DNA. In addition, we have identified a mechanism for involvement of Ku70 in activation of the CpG-DNA pathway.