OBJECTIVE: Atherosclerosis is driven by inflammatory reactions that are shared with the innate immune system. Toll-like receptor-9 (TLR9) is an intracellular pattern recognition receptor of the innate immune system that is currently under clinical investigation as a therapeutic target in inflammatory diseases. Here, we investigated whether TLR9 has a role in the development of atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. APPROACH AND RESULTS: Newly generated double-knockout ApoE(-/-):TLR9(-/-) mice and control ApoE(-/-) mice were fed a high-fat diet from 8 weeks and effects on lesion size, cellular composition, inflammatory status, and plasma lipids were assessed after 8, 12, 15, and 20 weeks. All 4 time points demonstrated exacerbated atherosclerotic lesion severity in ApoE(-/-):TLR9(-/-) mice, with a corresponding increase in lipid deposition and accumulation of macrophages, dendritic cells, and CD4(+) T cells. Although ApoE(-/-):TLR9(-/-) mice exhibited an increase in plasma very low-density lipoprotein/low-density-lipoprotein cholesterol, the very low-density lipoprotein/low-density lipoprotein:high-density lipoprotein ratio was unaltered because of a parallel increase in plasma high-density lipoprotein cholesterol. As a potential mechanism accounting for plaque progression in ApoE(-/-):TLR9(-/-) mice, CD4(+) T-cell accumulation was further investigated and depletion of these cells in ApoE(-/-):TLR9(-/-) mice significantly reduced lesion severity. As a final translational approach, administration of a TLR9 agonist (type B CpG oligodeoxynucleotide 1668) to ApoE(-/-) mice resulted in a reduction of lesion severity. CONCLUSIONS: Genetic deletion of the innate immune receptor TLR9 exacerbated atherosclerosis in ApoE(-/-) mice fed a high-fat diet. CD4(+) T cells were identified as potential mediators of this effect. A type B CpG oligodeoxynucleotide TLR9 agonist reduced lesion severity, thus identifying a novel therapeutic approach in atherosclerosis.