Immunostimulatory CpG motifs have been implicated as a major contributor to the acute inflammatory response associated with nonviral vectors, most prominently seen after systemic delivery of cationic lipid–plasmid DNA (pDNA) complexes. We have shown previously that complexes containing pDNA vectors that have been largely depleted of CpG motifs have significantly reduced acute toxicity when delivered systemically. However, several CpGs remain in these vectors and the toxicity is not negligible, especially at higher doses of complex. To determine the maximal reduction in the acute toxic response that could be achieved by eliminating CpG signaling, we injected cationic lipid–pDNA complexes into transgenic mice that are deficient in Toll-like receptor 9 (TLR9), which is the receptor that recognizes immunostimulatory CpG motifs. We observed significantly decreased adverse hematological changes and liver damage in TLR9^−/− mice compared to normal mice and increased survival at higher doses of complex. However, a pronounced loss of lymphocytes and platelets was still observed in the TLR9^−/− mice at higher doses. We also measured the toxicity in normal mice of systemically delivered complexes containing non-CpG oligonucleotides. Although serum transaminase levels were reduced, a loss of lymphocytes and platelets akin to that seen in the TLR9^−/− mice was observed. Taken together, these findings suggest that signaling through TLR9 contributes to the majority but not all of the toxic responses associated with systemic delivery of cationic lipid–pDNA complexes.