Background: Oxidative stress associated with cardiovascular disease (CVD) risk factors contributes to disease development. However, less is known whether specific subcellular components play a role in disease susceptibility. In this regard, it has been previously reported that vascular mitochondrial damage and dysfunction are associated with atherosclerosis. However, no studies have determined whether altered mitochondrial oxidant production directly influences atherogenic susceptibility and response in primary cells to atherogenic factors such as tumor necrosis factor-α (TNF-α). Objectives: We undertook this study to determine whether increased mitochondrial oxidant production affects atherosclerotic lesion development associated with CVD risk factor exposure and endothelial cell response to TNF-α. Methods: We assessed atherosclerotic lesion formation, oxidant stress, and mitochondrial DNA damage in male apolipoprotein E (apoE)-null mice with normal and decreased levels of mitochondrial superoxide dismutase-2 (SOD2; apoE-/- and apoE-/-, SOD2+/- respectively) exposed to environmental tobacco smoke or filtered air. Results: Atherogenesis, oxidative stress, and mitochondrial damage were significantly higher in apoE-/-, SOD2+/- mice than in apoE-/-, controls. Furthermore, experiments with small interfering RNA in endothelial cells revealed that decreased SOD2 activity increased TNF-α-mediated cellular oxidant levels compared with controls. Conclusions: Endogenous mitochondrial oxidative stress is an important CVD risk factor that can modulate atherogenesis and cytokine-induced endothelial cell oxidant generation. Consequently, CVD risk factors that induce mitochondrial damage alter cellular response to endogenous atherogenic factors, increasing disease susceptibility.