Neutrophils detect bacterial constituents, including bacterial DNA (CpG DNA), which elicits innate immunity and prolongs the functional life span of neutrophils through suppression of apoptosis. Both the anti-apoptotic protein Mcl-1 and activation of NF-κB have been implicated in neutrophil survival, but there is no evidence that these are linked in neutrophils. We hypothesized that CpG DNA could simultaneously activate these pathways. High purity CpG DNA (0.4-3.2 µg/ml) extended the life span of human neutrophils in vitro by delaying apoptosis through altering the rate of Mcl-1 turnover. CpG DNA slightly decreased Mcl-1 protein level in the presence of cyclohexmide and the proteasome inhibitor MG132 had little effect on Mcl-1 expression in CpG DNA-treated neutrophils. In contrast, CpG DNA evoked rapid increases in DNA binding by NF-κB/p65 and Mcl-1 mRNA. NF-κB inhibitors and the telomere-derived TLR9 inhibitory oligonucleotide 5'-TTT AGG GTT AGG GTT AGG G-3' markedly reduced Mcl-1 protein levels and subsequently abrogated suppression of apoptosis by CpG DNA. Furthermore, CpG DNA attenuated the decreases in Mcl-1 in both cell lysate and nucleus of neutrophils undergoing spontaneous apoptosis and increased Mcl-1 translocation to the mitochondria, leading to preservation of mitochondrial transmembrane potential. These results demonstrate that CpG DNA through toll-like receptor 9 links two survival signaling pathways by delaying apoptosis through induction of NF-κB-mediated Mcl-1 gene transcription and promoting Mcl-1 translocation to the mitochondria.