The brain regulates feeding by responding to dietary factors and metabolism. In obesity, obese individuals show disrupted energy homeostasis which is crucial for the survival and health of organisms to control hunger and energy balance. Although hypothalamic reactive astrocytes in lateral hypothalamic area (LHA) were observed in high fat diet-induced obese (DIO) mouse model, their role has been poorly understood. Here, we report immunohistochemical, electrophysiological data showing increased level of inhibitory gliotransmitter GABA in reactive astrocytes of DIO mice. We have previously demonstrated that monoamine oxidase-B (MAO-B) is responsible for the synthesis of GABA and Best1 channel for tonic release of GABA in reactive astrocytes in Alzheimer disease model mouse. When DIO mice were administered with a newly developed selective and reversible MAO-B inhibitor (KDS2010) dissolved in drinking water, they showed significantly reduced body mass compared to the groups fed with high fat diet (HFD) and drinking water only. However, total amount of food intake was similar between HFD with KDS2010 group and HFD with water group. These results imply that hypothalamic reactive astrocytes in LHA of the DIO model aberrantly and abundantly synthesize GABA via MAO-B and release via Best1. The astrocytic GABA may alter the activity of neurons for facilitating energy expenditure in DIO model. We propose that selective inhibition of astrocytic GABA synthesis or release may be a potential molecular target for treating obesity without compromising appetite.