Sirtuin 3 (Sirt3) is an NAD(+)-dependent mitochondrial deacetylase associated with superoxide dismutase 2 (SOD2)-mediated protection from oxidative stress. We have reported accelerated weight gain and impaired metabolic flexibility in atherosclerotic Sirt3 (-/-) mice. Oxidative stress is a hallmark of endothelial dysfunction. Yet, the role of Sirt3 in this context remains unknown. Thus, we aimed to unravel the effects of endogenous Sirt3 on endothelial function and oxidative stress. Knockdown of Sirt3 in human aortic endothelial cells (HAEC) increased intracellular mitochondrial superoxide accumulation, as assessed by electron spin resonance spectroscopy and fluorescence imaging. Endothelium-dependent relaxation of aortic rings from Sirt3 (-/-) mice exposed to a normal diet did not differ from wild-type controls. However, following 12 weeks of high-cholesterol diet and increasing oxidative stress, endothelial function of Sirt3 (-/-) mice was mildly impaired compared with wild-type controls. Relaxation was restored upon enhanced superoxide scavenging using pegylated superoxide dismutase. Knockdown of Sirt3 in cultured HAEC diminished SOD2 specific activity, which was compensated for by a CCAAT/enhancer binding protein beta (C/EBP-β)-dependent transcriptional induction of SOD2. Abrogation of this feedback regulation by simultaneous knockdown of C/EBP-β and Sirt3 exacerbated mitochondrial superoxide accumulation and culminated into endothelial cell death upon prolonged culture. Taken together, Sirt3 deficiency induces a mild, superoxide-dependent endothelial dysfunction in mice fed a high-cholesterol diet. In cultured endothelial cells, a novel C/EBP-β-dependent rescue mechanism maintains net SOD2 activity upon transient knockdown of Sirt3.