A serine/threonine protein kinase, Cot/Tpl2, is indispensable for extracellular signal–regulated kinase (ERK) activation and production of TNF-α and PGE₂ in LPS-stimulated macrophages. We show here that Cot/Tpl2 is also activated by other Toll-like receptor (TLR) ligands. Bacterial DNA rich in the dinucleotide CG (CpG-DNA), unlike LPS or synthetic lipopeptide, activated ERK in a Cot/Tpl2–independent manner. Peritoneal macrophages and bone marrow–derived DCs from Cot/Tpl2^–/– mice produced significantly more IL-12 in response to CpG-DNA than those from WT mice. Enhanced IL-12 production in Cot/Tpl2^–/– macrophages is, at least partly, regulated at the transcriptional level, and the elevated IL-12 mRNA level in Cot/Tpl2^–/– macrophages is accompanied by decreased amounts of IL-12 repressors, such as c-musculoaponeurotic fibrosarcoma (c-Maf) and GATA sequence in the IL-12 promoter–binding protein (GA-12–binding protein; GAP-12) in the nucleus. Consistently, Cot/Tpl2^–/– mice showed Th1-skewed antigen-specific immune responses upon OVA immunization and Leishmania major infection in vivo. These results indicate that Cot/Tpl2 is an important negative regulator of Th1-type adaptive immunity, that it achieves this regulation by inhibiting IL-12 production from accessory cells, and that it might be a potential target molecule in CpG-DNA–guided vaccination.