BACKGROUND—Mitochondrial DNA (mtDNA) damage occurs in both circulating cells and the vessel wall in human atherosclerosis. However, it is unclear whether mtDNA damage directly promotes atherogenesis or is a consequence of tissue damage, which cell types are involved, and whether its effects are mediated only through reactive oxygen species. METHODS AND RESULTS—mtDNA damage occurred early in the vessel wall in apolipoprotein E–null (ApoE) mice, before significant atherosclerosis developed. mtDNA defects were also identified in circulating monocytes and liver and were associated with mitochondrial dysfunction. To determine whether mtDNA damage directly promotes atherosclerosis, we studied ApoE mice deficient for mitochondrial polymerase-γ proofreading activity (polG/ApoE). polG/ApoE mice showed extensive mtDNA damage and defects in oxidative phosphorylation but no increase in reactive oxygen species. polG/ApoE mice showed increased atherosclerosis, associated with impaired proliferation and apoptosis of vascular smooth muscle cells, and hyperlipidemia. Transplantation with polG/ApoE bone marrow increased the features of plaque vulnerability, and polG/ApoE monocytes showed increased apoptosis and inflammatory cytokine release. To examine mtDNA damage in human atherosclerosis, we assessed mtDNA adducts in plaques and in leukocytes from patients who had undergone virtual histology intravascular ultrasound characterization of coronary plaques. Human atherosclerotic plaques showed increased mtDNA damage compared with normal vessels; in contrast, leukocyte mtDNA damage was associated with higher-risk plaques but not plaque burden. CONCLUSIONS—We show that mtDNA damage in vessel wall and circulating cells is widespread and causative and indicates higher risk in atherosclerosis. Protection against mtDNA damage and improvement of mitochondrial function are potential areas for new therapeutics.