Brainstem respiratory networks generate behaviorally relevant patterns of rhythmic neural activity driving respiratory motor output. Neural circuits with inherent rhythmogenic properties of the pre-Bötzinger complex (pre-BötC), a functionally specialized region in the ventrolateral medulla (VLM), are traditionally thought to be the main source of this rhythmic excitatory drive. Accumulating evidence supports the modulatory role of astrocytes, as chemosensory elements of the respiratory network, in regulating rhythm generation and respiratory motor output. Astrocytic release of gliotransmitter(s), mainly adenosine triphosphate (ATP), is shown to be responsible for astrocytic modulation of the respiratory network. Here, we report direct evidence demonstrating the active role of astrocytes in shaping rodent respiratory activity in vivo. Unilateral optogenetic stimulation of astrocytes expressing virally-transduced calcium translocating channelrhodopsin (CatCh) within the pre-BötC network in adult rats anaesthetized with urethane (1.3 mg • kg -1, i.p., n=5) significantly increased the amplitude of phrenic nerve inspiratory activity by 36.54±13.26% when compared to the control group (n=4, student t-test, p<0.004). This result demonstrates a potential role for astrocytes in shaping the activity of pre-BötC neuronal circuits.