Sustained hypoxia (SH) produces cardiovascular and respiratory changes in unacclimatized humans ascending to high altitude due to peripheral chemoreflex activation. Moreover, hypoxia also produces changes in structural and functional integrity of glial cells, which are essential for neuronal activity. In this study we evaluated the effect of short-term SH (24 hours, pO210%) on the neuron-glia interaction at the NTS, the first synaptic station of peripheral chemoreflex in the brainstem. Using brainstem slices and whole-cell patch clamp, we observed that passive properties of NTS astrocytes were not affected by SH [input resistance: control: 44.5 ± 14 MΩ (n=8) vs SH: 31.5 ± 10 MΩ (n=4)][RMP control: -84 ± 3.6 mV (n=10) vs SH: -75 ± 5.2 mV (n=7)]. Since protease-activated receptors 1 (PAR1) are found only in NTS astrocytes and not in neurons, we also evaluated the glial modulation on neurotransmission by activation of astrocytes with SFLLRN-NH2 [PAR1 agonist (20 μM)]. SFLLRN-NH2 decreased the amplitude of evoked excitatory post-synaptic currents (eEPSCs) in NTS neurons from SH animals to levels similar to that observed in control group [control: -51 ± 5 pA (n=3) vs SH: -56 ± 8 pA (n=5)]. SFLLRN-NH2 did not change the RMP of NTS neurons in both groups [control: aCSF: -65.5 ± 1.5 mV vs aCSF + SFLLRN-NH2: -61 ± 5 mV, (n=4); SH: aCSF: -63 ± 5 vs aCSF + SFLLRN-NH2: -56 ± 8 mV, (n=4)]. In addition, using in vivo labeling and multi-photon microscopy, we observed that SH decreased the cellular density of astrocytes in NTS [control: 0.3 ± 0.004 (n=5) vs SH: 0.1 ± 0.01 cells / μm2 (n=5)]. Recording intracellular calcium concentration (calcium waves), we observed that SH reduced the astrocytic response to another hypoxia exposure [Average of fluorescence alterations (ΔF/F) to 5 min exposure to hypoxic solution – control: 43% vs SH: 3%]. We conclude that SH decreased the number of astrocytes as well their inhibitory modulation on excitatory transmission on the NTS neurons, which may contribute to the enhancement of baro- and chemoreflex responses observed in this experimental model.