It has been known since the discovery of DNA vaccines > 20 y ago that DNA vaccines can function as adjuvants. Our recent study reported the involvement of Aim2 as the sensor of DNA vaccines in eliciting Ag-specific Ab responses. Our findings indicated the presence of previously unrecognized innate immune response pathways in addition to the TLR9 pathway, which is mainly activated by the CpG motifs of DNA vaccines. Our data further demonstrated the requirement of type I IFN in DNA vaccine-induced immune responses via the Aim2 pathway, but the exact downstream molecular mechanism was not characterized. In the present study, we investigated the roles of the putative DNA sensor cyclic GMP-AMP synthase (cGas), as well as the downstream IFN regulatory factors (IRF) 3 and 7 in type I IFN induction and Ag-specific immune responses elicited by DNA vaccination. Our results showed that DNA vaccine-induced, Irf7-dependent signaling, as part of the Sting pathway, was critical for generation of both innate cytokine signaling and Ag-specific B and T cell responses. In contrast, Irf3 was not as critical as expected in this pathway and, more surprisingly, immune responses elicited by DNA vaccines were not cGas-dependent in vivo. Data from this study provide more details on the innate immune mechanisms involved in DNA vaccination and further enrich our understanding on the potential utility of DNA vaccines in generating Ag-specific immune responses.