Central chemoreception is essential for adjusting breathing to physiological demands, and for maintaining CO2 and pH homeostasis in the brain. CO2-induced ATP release from brainstem astrocytes stimulates breathing. NMDA receptor (NMDAR) antagonism reduces the CO2-induced hyperventilation by unknown mechanisms. Here we show that astrocytes in the mouse caudal medullary brainstem can synthesize, store, and release D-serine, an agonist for the glycine-binding site of the NMDAR, in response to elevated CO2 levels. We show that systemic and raphe nucleus D-serine administration to awake, unrestrained mice increases the respiratory frequency. Application of D-serine to brainstem slices also increases respiratory frequency, which was prevented by NMDAR blockade. Inhibition of D-serine synthesis, enzymatic degradation of D-serine, or the sodium fluoroacetate-induced impairment of astrocyte functions decrease the basal respiratory frequency and the CO2-induced respiratory response in vivo and in vitro. Our findings suggest that astrocytic release of D-serine may account for the glutamatergic contribution to central chemoreception.Astrocytes are involved in chemoreception in brainstem areas that regulate breathing rhythm, and astrocytes are known to release D-serine. Here the authors show that astrocyte release of D-serine contributes to CO2 sensing and breathing in brainstem slices, and in vivo in awake unrestrained mice.