Neuron-glia communication in the central nervous system is emerging as an important component of neuronal signaling in respiratory control centers of the brainstem. To investigate glial cell contributions to ventilatory acclimatization to hypoxia, I measured the time course of activation of microglia and astrocytes’ neural circuits that control breathing following different lengths of exposure to sustained hypoxia (10% O2). Rats were exposed to sustained hypoxia at the following time points: 30 minutes, 1-, 4-, and 24-hours, 4- and 7-days. Activation of glial cells was measured in two respiratory control centers: the nucleus tractus solitarius (NTS), a sensory integrative center known to be important for ventilatory acclimatization to hypoxia, and the hypoglossal (XII) motor nucleus, a respiratory motor nucleus adjacent to the NTS. Microglia (Iba-1 positive cells) show an active morphology (shorter and fewer branches measured with image analysis, IMARIS) after 1 hour of hypoxia in the NTS and XII and return to the resting ramified state (longer filaments and extensive branching) after 4 hours of hypoxia. The number of microglia stayed constant throughout activation. Astrocytes (glial fibrillary acidic protein, GFAP positive cells) increased GFAP antibody immunofluorescent intensity after 4 hours of sustained hypoxia in the NTS but not until 7 days in XII. Astrocyte proliferation was only observed after 7 days of sustained hypoxia in the hypoglossal motor nucleus. These results show that glial activation with sustained hypoxia occurs sequentially with microglia activation preceding astrocyte activation and a time course that is unique in different medullary respiratory centers. This indicates an important role for glia in neural plasticity with sustained hypoxia and ventilatory acclimatization to hypoxia.