8-Hydroxy-2-deoxyguanosine (8-OHdG), a marker of oxidative stress, has been recently rediscovered to inhibit Rac1 in neutrophils and macrophages, thereby inhibiting Rac1-linked functions of these cells, including reactive oxygen species production through NADPH oxidase activation, phagocytosis, chemotaxis, and cytokine release. In vascular smooth muscle cells (VSMCs), reactive oxygen species also induce abnormal proliferation and migration leading to progression of atherosclerosis. Based upon the involvement of reactive oxygen species in phagocytic cells and VSMCs during the atherosclerotic process, we hypothesized that 8-OHdG could have antiatherosclerotic action and tested this hypothesis in an experimentally induced atherosclerosis in mice. Partially ligated ApoE knockout mice, a more physiologically relevant model of low and oscillatory flow, developed an advanced lesion in 2 weeks, and orally administered 8-OHdG significantly reduced plaque formation along with reduced superoxide formation, monocyte/macrophage infiltration, and extracellular matrix (ECM) accumulation. The effects of 8-OHdG observed in primary VSMCs were consistent with the in vivo effects of 8-OHdG and were inhibitory to angiotensin II or platelet-derived growth factor-induced production of reactive oxygen species, proliferation, migration, and ECM production. Also, angiotensin II-induced Rac1 activity in VSMCs was significantly inhibited by 8-OHdG, and transfection of constitutively active Rac1 reversed the inhibitory effect of 8-OHdG on VSMC activation. Molecular docking study showed that 8-OHdG stabilizes Rac1-GEF complex, indicating the physical contact of 8-OHdG with Rac1. These findings highly suggest that the antiatherosclerotic effect of 8-OHdG is mediated by inhibition of Rac1 activity. In conclusion, our results show a novel action of orally active 8-OHdG in suppressing atherosclerotic plaque formation in vivo and VSMC activation in vitro through inhibition of Rac1, which emphasizes a new therapeutic avenue to benefit atherosclerosis.