The effects of obesity on the lateral hypothalamic area (LHA; a highly conserved brain region that mediates feeding behaviour) have been unclear to date. In this study, a combination of high-throughput single-cell RNA sequencing and longitudinal in vivo two-photon calcium imaging revealed that, in mice, glutamatergic neurons in the LHA act as a brake on feeding and supresses further food intake. Notably, the activity of these neurons is affected by diet-induced obesity, promoting further overeating.