NakedDNA vaccines given by intramuscular injection are efficient in mouse models, but they require improvement for human use. As the immunogenicity ofDNA vaccines depends, to a large extent, on the presence ofCpG motifs as built-in adjuvants, we addressed this issue by inserting three types of humanCpG motifs (A-type, B-type, andC-type) into the backbone of nonviralDNA and viralDNA replicon vectors with distinct immunostimulatory activities on human PBMCs. The adjuvant effects ofCpGmodifications inDNA vaccines expressing three types of antigens (β-Gal, AHc, or PA4) were then characterized in mice and found to significantly enhance antigen-specific humoral and cell-mediated immune responses. The three types ofCpG motifs also differentially affected and modulated immune responses and protective potency against botulinum neurotoxin serotypeA andBacillus anthracisA16R challenge. Taken together, these results demonstrate that insertion of humanCpG motifs can differentially modulate the immunogenicity of nonviralDNA vaccines as well as viralDNA replicon vaccines. Our study provides not only a better understanding of thein vivo activities ofCpG motif adjuvants but implications for the rational design of such motifs as built-in adjuvants forDNA vectors targeting specific antigens.