Oxygen delivery by Hb is essential for vertebrate life. Three amino acids in Hb are strictly conserved in all mammals and birds, but only two of those, a His and a Phe that stabilize the heme moiety, are needed to carry O ₂. The third conserved residue is a Cys within the β-chain (βCys93) that has been assigned a role in S-nitrosothiol (SNO)-based hypoxic vasodilation by RBCs. Under this model, the delivery of SNO-based NO bioactivity by Hb redefines the respiratory cycle as a triune system (NO/O ₂/CO ₂). However, the physiological ramifications of RBC-mediated vasodilation are unknown, and the apparently essential nature of βCys93 remains unclear. Here we report that mice with a βCys93Ala mutation are deficient in hypoxic vasodilation that governs blood flow autoregulation, the classic physiological mechanism that controls tissue oxygenation but whose molecular basis has been a longstanding mystery. Peripheral blood flow and tissue oxygenation are decreased at baseline in mutant animals and decline excessively during hypoxia. In addition, βCys93Ala mutation results in myocardial ischemia under basal normoxic conditions and in acute cardiac decompensation and enhanced mortality during transient hypoxia. Fetal viability is diminished also. Thus, βCys93-derived SNO bioactivity is essential for tissue oxygenation by RBCs within the respiratory cycle that is required for both normal cardiovascular function and circulatory adaptation to hypoxia. Significance Oxygen delivery by RBC Hb is essential for life. Just three amino acids in Hb are conserved in all mammals and birds, but only two of those are required to carry oxygen. The third, a Cys within the β-chain, βCys93, has been assigned a role in carrying nitric oxide, which mediates vasodilation. However, the physiological importance of RBC-mediated vasoregulation is unknown. We show that blood flow and tissue oxygenation are markedly impaired in mice with a βCys93Ala mutation. The βCys93Ala mutation also results in myocardial ischemia, cardiac decompensation, and enhanced mortality. These findings support a new view of the respiratory cycle wherein, remarkably, RBCs regulate blood flow and (βCys93NO)-Hb is necessary for adequate tissue oxygenation and normal cardiovascular function.