Abstract:  Glial fibrillary acidic protein (GFAP)‐expressing cells (GFAP⁺ glial cells) are the predominant cell type in the central and peripheral nervous systems. Our understanding of the role of GFAP⁺ glial cells and their signalling systems in vivo is limited due to our inability to manipulate these cells and their receptors in a cell type‐specific and non‐invasive manner. To circumvent this limitation, we developed a transgenic mouse line (GFAP‐hM3Dq mice) that expresses an engineered G_q protein‐coupled receptor (G_q‐GPCR) known as hM3Dq DREADD (designer receptor exclusively activated by designer drug) selectively in GFAP⁺ glial cells. The hM3Dq receptor is activated solely by a pharmacologically inert, but bioavailable, ligand (clozapine‐N‐oxide; CNO), while being non‐responsive to endogenous GPCR ligands. In GFAP‐hM3Dq mice, CNO administration increased heart rate, blood pressure and saliva formation, as well as decreased body temperature, parameters that are controlled by the autonomic nervous system (ANS). Additionally, changes in activity‐related behaviour and motor coordination were observed following CNO administration. Genetically blocking inositol 1,4,5‐trisphosphate (IP₃)‐dependent Ca²⁺ increases in astrocytes failed to interfere with CNO‐mediated changes in ANS function, locomotor activity or motor coordination. Our findings reveal an unexpectedly broad role of GFAP⁺ glial cells in modulating complex physiology and behaviour in vivo and suggest that these effects are not dependent on IP₃‐dependent increases in astrocytic Ca²⁺.