Although DNA vaccines are already in use for treatment of some animal diseases, they suffer from lower immunogenicity in humans which limits their effectiveness. Thus, recent studies have been focused on strategies to improve the immunogenicity of DNA vaccines. However, there is little known about the molecular and immunological mechanisms by which DNA vaccines work. It has long been the central dogma that DNA vaccine immunogenicity can be attributed to its immunostimulatory CpG motifs acting as 'a built-in adjuvant", which is recognized by Toll-like receptor (TLR) 9, the sole receptor for CpG motifs. Recent research, however, has provided evidence for a new mechanism of action for DNA vaccines. It was reported that the adjuvant effect of plasmid DNA is mediated by its double-stranded structure, which activates TBK1-dependent innate immune signaling pathways in the absence of TLRs. Moreover, TBK1-signaling may delineate direct or indirect (cross) antigen presentation through distinct types of cells in vivo, critical for the induction of antigen-specific CD4(+) or CD8(+) T cells, respectively. This additional information about the mechanism of action of DNA vaccines will lead to improvements in their efficacy and safety.