Nitric oxide (NO ^• ) competitively inhibits oxygen consumption by mitochondria at cytochrome c oxidase and S -nitrosates thiol proteins. We developed mitochondria-targeted S -nitrosothiols (MitoSNOs) that selectively modulate and protect mitochondrial function. The exemplar MitoSNO1, produced by covalently linking an S -nitrosothiol to the lipophilic triphenylphosphonium cation, was rapidly and extensively accumulated within mitochondria, driven by the membrane potential, where it generated NO ^• and S -nitrosated thiol proteins. MitoSNO1-induced NO ^• production reversibly inhibited respiration at cytochrome c oxidase and increased extracellular oxygen concentration under hypoxic conditions. MitoSNO1 also caused vasorelaxation due to its NO ^• generation. Infusion of MitoSNO1 during reperfusion was protective against heart ischemia-reperfusion injury, consistent with a functional modification of mitochondrial proteins, such as complex I, following S -nitrosation. These results support the idea that selectively targeting NO ^• donors to mitochondria is an effective strategy to reversibly modulate respiration and to protect mitochondria against ischemia-reperfusion injury.