Administration of an excess of oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG-S ODNs) may induce systemic inflammatory response syndrome (SIRS) and sepsis. Therefore, it is important to develop neutralizing CpG ODNs (CpG-N ODNs), which can be used to reduce the release of cytokines induced by the presence of CpG-S ODNs. In the present study, CpG-N ODN208 (5'-TGCCGCGGCAGA-3'), a neutralizing twelve-oligodeoxynucleotide molecule recently identified in our laboratory, inhibited TNF-alpha release from human peripheral blood mononuclear cells (hPBMCs) and murine RAW264.7 cells induced by CpG-S ODN exposure in a dose- and time-dependent manner. Flow cytometry revealed that CpG-N ODN208 decreased cell-surface binding and internalization of 6-FAM-CpG-S ODN. However, the decreased cell-surface binding and internalization of CpG-S ODN could not completely account for the decreased TNF-alpha release. RT-PCR experiments revealed that CpG-N ODN treatment could down-regulate the CpG-S ODN-induced upregulation of Toll-like receptor 9 (TLR9) mRNA expression. This finding suggested that the decreased cytokine release following CpG-N ODN treatment might be related to decreased TLR9 mRNA expression. In in vivo experiments, no protection was found when the ratio of CpG-N ODN to CpG-S ODN delivered to mice was 3:1. However, at a 5:1 ratio, CpG-N ODN208 could protect mice from an ordinarily lethal dose of CpG-S ODN. Furthermore, we found that CpG-N ODN208 treatment decreased serum TNF-alpha levels in mice injected with sublethal doses of CpG-S ODN whether the CpG-N ODN208 was added prior to or concurrent with the CpG-S ODN. Our results demonstrated that CpG-N ODN-mediated protection against a lethal challenge by CpG-S ODN was associated with the reduction of TNF-alpha release.