AbstractBrain glucose-sensing neurons detect glucose fluctuations and prevent severe hypoglycemia, but mechanisms mediating functions of these glucose-sensing neurons are unclear. Here we report that estrogen receptor-α (ERα)-expressing neurons in the ventrolateral subdivision of the ventromedial hypothalamic nucleus (vlVMH) can sense glucose fluctuations, being glucose-inhibited neurons (GI-ERα^vlVMH) or glucose-excited neurons (GE-ERα^vlVMH). Hypoglycemia activates GI-ERα^vlVMH neurons via the anoctamin 4 channel, and inhibits GE-ERα^vlVMH neurons through opening the ATP-sensitive potassium channel. Further, we show that GI-ERα^vlVMH neurons preferentially project to the medioposterior arcuate nucleus of the hypothalamus (mpARH) and GE-ERα^vlVMH neurons preferentially project to the dorsal Raphe nuclei (DRN). Activation of ERα^vlVMH to mpARH circuit and inhibition of ERα^vlVMH to DRN circuit both increase blood glucose. Thus, our results indicate that ERα^vlVMH neurons detect glucose fluctuations and prevent severe hypoglycemia in mice.