Parallel to attenuated and subunit vaccines, DNA vaccines require adjuvant signals in addition to antigen presentation for the induction of adaptive immune responses. As opposed to common beliefs, increasing evidence is showing that Toll-like receptor 9 activation by CpG motifs present in DNA vaccines are not vital for the induction of immune responses in vivo. Investigations on the signaling pathways of the adjuvant effect mediated by DNA vaccines have revealed other important mediators. DNA-dependent activator of interferon regulatory factors (DAI) and absent in melanoma (AIM)2 were recently identified as cytosolic DNA sensors that respond with the release of type I interferon and proinflammatory cytokines. Both are distinct molecules with different signaling pathways. AIM2 acts through inflammasomes to activate caspase-1, whilst DAI activates the transcription factors, NF-kappaB and interferon regulatory factor 3. Most significantly, the noncanonical IkappaB kinase, TANK-binding kinase-1, was identified as the essential signaling component in DNA vaccines that is responsible for the generation of immune responses. This review provides an update on the cellular detection and the subsequent signaling pathways mediated by DNA vaccines.