Information transfer within neuronal circuits depends on the balance and recurrent activity of excitatory and inhibitory neurotransmission. Chloride (Cl -) is the major central nervous system (CNS) anion mediating inhibitory neurotransmission. Astrocytes are key homoeostatic glial cells populating the CNS, although the role of these cells in regulating excitatory-inhibitory balance remains unexplored. Here we show that astrocytes act as a dynamic Cl - reservoir regulating Cl - homoeostasis in the CNS. We found that intracellular chloride concentration ([Cl -] i) in astrocytes is high and stable during sleep. In awake mice astrocytic [Cl -] i is lower and exhibits large fluctuation in response to both sensory input and motor activity. Optogenetic manipulation of astrocytic [Cl -] i directly modulates neuronal activity during locomotion or whisker stimulation. Astrocytes thus serve as a dynamic source of extracellular Cl - available for GABAergic transmission in awake mice, which represents a mechanism for modulation of the inhibitory tone during sustained neuronal activity.