Odontoblasts are the first-line defense cells against invading microorganisms. Toll-like receptors (TLRs) play a crucial role in innate immunity, and TLR9 is involved in the recognition of microbial DNA. This study aimed to investigate whether odontoblasts can respond to CpG DNA and to determine the intracellular signaling pathways triggered by CpG DNA. We found that the mouse odontoblast-like cell line MDPC-23 constitutively expressed TLR9. Exposure to CpG ODN induced a potent proinflammatory response based on an increase of IL-6 and TNF-alpha expression. Pretreatment with an inhibitory MyD88 peptide or a specific inhibitor for TLR9, NF-kappa B or I kappa B alpha markedly inhibited CpG ODN-induced IL-6 and TNF-alpha expression. Moreover, the CpG ODN-mediated increase of kappa B-luciferase activity in MDPC-23 cells was suppressed by the overexpression of dominant negative mutants of TLR9. MyD88 and I kappa B alpha, but not by the dominant negative mutant of TLR4. This result suggests a possible role for the CpG DNA-mediated immune response in odontoblasts and indicates that TLR9, MyD88 and NF-kappa B are involved in this process. Crown Copyright (C) 2010 Published by Elsevier Inc. All rights reserved.