Mycobacterium consists up to 7% of mycobacterial DNA-binding protein 1 (MDP1) in total cellular proteins. Host immune responses to MDP1 were studied in mice to explore the antigenic properties of this protein. Anti-MDP1 IgG was produced after infection with either bacillus Calmette-Guerin or Mycobacterium tuberculosis in C3H/HeJ mice. However, the level of Ab was remarkably low when purified MDPI was injected. MDPI is considered to be associated with DNA in nucleoid, which contains immunostimulatory CpG motif. Therefore, we examined coadministration of MDPI and DNA derived from M. tuberculosis. Consequently, this procedure significantly enhanced the production of MDP1-specific IgG. Five nanograms of DNA was enough to enhance MDP1-specific IgG production in the administration of 5 mu g of MDP1 into mice. Strong immune stimulation by such a small amount of DNA is noteworthy, because > 1,000- to 100,000-fold doses of CpG DNAs are used for immune activation. A synthetic peptide-based study showed that B cell epitopes were different between mice administered MDPI alone and those given a mixture of MDP1 and DNA, suggesting that DNA alters the three-dimensional structure of MDP1. Coadministration of DNA also enhanced MDP1-specific IFN-gamma production and reduced the bacterial burden of a following challenge of M. tuberculosis, showing that MDP1 is a novel vaccine target. Finally, we found that MDP1 remarkably enhanced TLR9-dependent immune stimulation by unmethylated CpG oligo DNA in vitro. To our knowledge, MDPI is the first protein discovered that remarkably augments the CpG-mediated immune response and is a potential adjuvant for CpG DNA-based immune therapies.