Unmethylated CpG DNA binds to the Toll-like receptor 9 (TLR9) and activates NF-κB to induce cytokine genes in dendritic cells (DCs). IFN regulatory factor (IRF)-8/IFN consensus sequence binding protein is a transcription factor important for development and activation of DCs. We found that DCs from IRF-8−/− mice were unresponsive to CpG and failed to induce TNF-α and IL-6, targets of NF-κB. Revealing a signaling defect selective for CpG, these cytokines were robustly induced in IRF-8−/− DCs in response to LPS that signals through TLR4. IRF-8−/− DCs expressed TLR9, adaptor myeloid differentiation factor 88, and other signaling molecules, but CpG failed to activate NF-κB in−/− cells. This was due to the selective inability of−/− DCs to activate I-κB kinase αβ, the kinases required for NF-κB in response to CpG. IRF-8 reintroduction fully restored CpG activation of NF-κB and cytokine induction in−/− DCs. Together, TLR signals that activate NF-κB are diverse among different TLRs, and TLR9 signaling uniquely depends on IRF-8 in DCs.