Background Toll‐like receptor ( TLR ) 9 recognizes bacterial DNA , activating innate immunity, whereas it also provokes inflammation in response to fragmented DNA released from mammalian cells. We investigated whether TLR 9 contributes to the development of vascular inflammation and atherogenesis using apolipoprotein E–deficient ( Apoe −/− ) mice. Methods and Results Tlr9 ‐deficient Apoe −/− ( Tlr9 −/− Apoe −/− ) mice and Apoe −/− mice on a Western‐type diet received subcutaneous angiotensin II infusion (1000 ng/kg per minute) for 28 days. Angiotensin II increased the plasma level of double‐stranded DNA, an endogenous ligand of TLR 9, in these mice. Genetic deletion or pharmacologic blockade of TLR 9 in angiotensin II–infused Apoe −/− mice attenuated atherogenesis in the aortic arch ( P RNA expression of inflammatory molecules in the aorta with no alteration of metabolic parameters. On the other hand, restoration of TLR 9 in bone marrow in Tlr9 −/− Apoe −/− mice promoted atherogenesis in the aortic arch ( P TLR 9 agonist markedly promoted proinflammatory activation of Apoe −/− macrophages, partially through p38 mitogen‐activated protein kinase signaling. In addition, genomic DNA extracted from macrophages promoted inflammatory molecule expression more effectively in Apoe −/− macrophages than in Tlr9 −/− Apoe −/− macrophages. Furthermore, in humans, circulating double‐stranded DNA in the coronary artery positively correlated with inflammatory features of coronary plaques determined by optical coherence tomography in patients with acute myocardial infarction ( P Conclusions TLR 9 plays a pivotal role in the development of vascular inflammation and atherogenesis through proinflammatory activation of macrophages. TLR 9 may serve as a potential therapeutic target for atherosclerosis.