Acute cardiorespiratory responses to O₂ deficiency are essential for physiological homeostasis. The prototypical acute O₂-sensing organ is the carotid body, which contains glomus cells expressing K⁺ channels whose inhibition by hypoxia leads to transmitter release and activation of nerve fibers terminating in the brainstem respiratory center. The mechanism by which changes in O₂ tension modulate ion channels has remained elusive. Glomus cells express genes encoding HIF2α (Epas1) and atypical mitochondrial subunits at high levels, and mitochondrial NADH and reactive oxygen species (ROS) accumulation during hypoxia provides the signal that regulates ion channels. We report that inactivation of Epas1 in adult mice resulted in selective abolition of glomus cell responsiveness to acute hypoxia and the hypoxic ventilatory response. Epas1 deficiency led to the decreased expression of atypical mitochondrial subunits in the carotid body, and genetic deletion of Cox4i2 mimicked the defective hypoxic responses of Epas1-null mice. These findings provide a mechanistic explanation for the acute O₂ regulation of breathing, reveal an unanticipated role of HIF2α, and link acute and chronic adaptive responses to hypoxia.