Oxysterols are metabolites of cholesterol produced in peripheral tissues to eliminate cholesterol. 27-hydroxycholesterol (27HC) is the most abundant oxysterol and can cross the blood-brain barrier. Interestingly, 27HC has been identified as an endogenous selective estrogen receptor modulator (SERM) for both estrogen receptor α and β (ERα/β). Considering the regulatory effects of brain estrogen/ERα signaling on energy metabolism, we hypothesize that the 27HC binds with ERα in the arcuate nucleus of the hypothalamus (ARH) of the brain to modulate energy homeostasis. In supporting this, we found that a single acute intracerebroventricular injection of 27HC inhibited food intake in both male and female mice. This anorexigenic effect was also associated with the increased c-fos expression in the pro-opiomelanocortin neurons in ARH (POMCARH). Using brain slice patch-clamp recording, we consistently showed that 27HC dose-dependently actives POMCARH neurons in an ERα-dependent manner, suggesting a mediating role of ERα expressed by POMCARH neurons. Notably, we further revealed that the inhibitory effects of 27HC on food intake were blocked by pharmacological blockage or POMC-specific deletion of ERα. Consistently, chemogenetic inhibition of POMCARH neurons also blunted the anorexigenic effects of 27HC in mice. Mechanistically, we also demonstrated that the small conductance of the calcium-activated potassium (SK) channel is necessary for mediating the stimulatory effects of 27HC on POMCARH neurons. Therefore, our results support a model that 27HC acutely inhibits food intake by acting on ERα to stimulate POMCARH neuronal activity via inhibiting SK current. This 27HC/ERα/SK/POMC signaling pathway may serve as a critical defending mechanism against high-fat diet-induced obesity. These findings provide important insights into the role of cholesterol metabolites in the regulation of energy homeostasis and suggest potential therapeutic targets for the treatment of obesity and related metabolic disorders.