Disruption of microvascular blood flow is a common cause of tissue hypoxia in disease, yet no therapies are available that directly target the microvasculature to improve tissue oxygenation. Red blood cells (RBCs) autoregulate blood flow through S-nitroso-hemoglobin (SNO-Hb)-mediated export of nitric oxide (NO) bioactivity. We therefore tested the idea that pharmacological enhancement of RBCs using the S-nitrosylating agent ethyl nitrite (ENO) may provide a novel approach to improve tissue oxygenation. Serial ENO dosing was carried out in sheep (1-400 ppm) and humans (1-100 ppm) at normoxia and at reduced fraction of inspired oxygen (FiO2 ). ENO increased RBC SNO-Hb levels, corrected hypoxia-induced deficits in tissue oxygenation, and improved measures of oxygen utilization in both species. No adverse effects or safety concerns were identified. Inasmuch as impaired oxygenation is a major cause of morbidity and mortality, ENO may have widespread therapeutic utility, providing a first-in-class agent targeting the microvasculature.