The tight coupling between cerebral blood flow and neural activity is a key feature of normal brain function and forms the basis of functional hyperemia. The mechanisms coupling neural activity to vascular responses, however, remain elusive despite decades of research. Recent studies have shown that cerebral functional hyperemia begins in capillaries, and red blood cells (RBCs) act as autonomous regulators of brain capillary perfusion. RBCs then respond to local changes of oxygen tension (PO2) and regulate their capillary velocity. Using ex vivo microfluidics and in vivo two-photon microscopy, we examined RBC capillary velocity as a function of PO2 and showed that deoxygenated hemoglobin and band 3 interactions on RBC membrane are the molecular switch that responds to local PO2 changes and controls RBC capillary velocity. Capillary hyperemia can be controlled by manipulating RBC properties independent of the neurovascular unit, providing an effective strategy to treat or prevent impaired functional hyperemia