Synaptic inhibition is based on both tonic and phasic release of the inhibitory transmitter γ-aminobutyric acid (GABA). Although phasic GABA release arises from Ca(2+)-dependent exocytosis from neurons, the mechanism of tonic GABA release is unclear. Here we report that tonic inhibition in the cerebellum is due to GABA being released from glial cells by permeation through the Bestrophin 1 (Best1) anion channel. We demonstrate that GABA directly permeates through Best1 to yield GABA release and that tonic inhibition is eliminated by silencing of Best1. Glial cells express both GABA and Best1, and selective expression of Best1 in glial cells, after preventing general expression of Best1, fully rescues tonic inhibition. Our results identify a molecular mechanism for tonic inhibition and establish a role for interactions between glia and neurons in mediating tonic inhibition.