Significance Vascular oxidative stress and endothelial cell dysfunction contribute to various human pathologies. Here, we show that, in vascular endothelial cells, a key mitochondrial antioxidant enzyme, namely thioredoxin reductase 2 (TrxR2), plays a central role in the control of vascular integrity. Its targeted loss is associated with disruption of both nitric oxide (·NO) and redox homeostasis in vivo. Impaired TrxR2 activity leads to elevated steady-state levels of peroxynitrite, reflecting the decreased bioavailability of ·NO by its O ₂ ^{· −} mediated oxidative inactivation. Thus, TrxR2 represents a central control point of peroxynitrite levels by providing reducing equivalents to mitochondrial peroxiredoxins that, in turn, catalytically reduce peroxynitrite to nitrite. The data additionally support pharmacological approaches for mitochondrial peroxynitrite detoxification under conditions of vascular oxidative stress.