A potent immunostimulatory effect of DNA containing an unmethylated CpG motif was found in the course of research on water-soluble components from BCG possessing antitumor activity. Because such CpG motifs are relatively common in bacterial DNA, but rare in mammalian animal and plant DNA, they may be evolutionary adaptation augmenting innate immunity, most likely in response to pathogens that replicate within the host cells, such as viruses and intracellular bacteria. Microbial infection induces innate immunity by triggering pattern-recognition system. The infected cells produce proinflammatory cytokines that directly combat microbial invaders and express costimulating surface molecules, which develop adaptive immunity by inducing distinct T cell differentiation. Bacterial DNA with unmethylated CpG-DNA stimulates vertebrate immature immune cells to induce maturation and to produce Thl cytokines as well as TNF-alpha. Therefore, CpG-DNA functions as an adjuvant for regulating the initiation of Th1 differentiation. DNA vaccine including genes encoding mycobacterial proteins either MPB64 or HSP65 was assessed the ability to prevent the growth of bacilli in the lungs and spleens of guinea pigs after pulmonary challenge of virulent Mycobacterium tuberculosis H37Rv. Immunization with two constructs such as MPB64 and HSP65 elicited protective responses compared to a vector control or saline control. The roles of immunostimulatory CpG motifs in DNA vaccine developments and therapeutic applications have been discussed.