Liposome:DNA is a promising gene therapy vector. However, this vector can elicit a systemic inflammatory response syndrome (SIRS). Prior reports indicate that liposome:DNA vectors activate Toll-like receptor (TLR)9. We hypothesized that liposome:DNA vectors also activate the cytosolic DNA-sensing pathway, which signals via interferon (IFN) regulatory factor (IRF)3. To test this, we treated dendritic cells (DCs) with liposome:DNA in vitro and found that IRF3 was phosphorylated independent of TLR9. To test the contribution of this pathway in vivo, we injected a liposome:DNA vector into wild-type (WT), TLR9-knockout (KO), IRF3-KO, and TLR9-IRF3-double-KO (DKO) mice. WT mice exhibited a systemic inflammatory response, evidenced by elevations in serum cytokines, serum enzyme changes indicating organ damage, hypothermia, and mortality. The cytokine response was reduced in TLR9-KO, IRF3-KO, and TLR9-IRF3-DKO mice and all three groups survived. We found that IFN-gamma-KO mice that receive liposome:DNA had a reduced cytokine response and 100% survival. CD11c(+) and NK1.1(+) cells produced IFN-gamma and depleting CD11c(+) cells reduced the cytokine response in mice injected with liposome:DNA. These findings may facilitate the development of immunologically inert gene therapy vectors and may provide general insight into the mechanisms of SIRS.