GABA is the major inhibitory transmitter in the brain and is released not only from a subset of neurons but also from glia. Although neuronal GABA is well known to be synthesized by glutamic acid decarboxylase (GAD), the source of glial GABA is unknown. After estimating the concentration of GABA in Bergmann glia to be around 5–10�mm by immunogold electron microscopy, we demonstrate that GABA production in glia requires MAOB, a key enzyme in the putrescine degradation pathway. In cultured cerebellar glia, both Ca²⁺‐induced and tonic GABA release are significantly reduced by both gene silencing of MAOB and the MAOB inhibitor selegiline. In the cerebellum and striatum of adult mice, general gene silencing, knock out of MAOB or selegiline treatment resulted in elimination of tonic GABA currents recorded from granule neurons and medium spiny neurons. Glial‐specific rescue of MAOB resulted in complete rescue of tonic GABA currents. Our results identify MAOB as a key synthesizing enzyme of glial GABA, which is released via bestrophin 1 (Best1) channel to mediate tonic inhibition in the brain.