Estrogen receptor α (ERα) plays a pivotal role in the regulation of food intake and energy expenditure by estrogens. Although it is well documented that a disruption of ERα signaling in ERα knockout (ERKO) mice leads to an obese phenotype, the sites of estrogen action and mechanisms underlying this phenomenon are still largely unknown. In the present study, we exploited RNA interference mediated by adeno-associated viral vectors to achieve focused silencing of ERα in the ventromedial nucleus of the hypothalamus, a key center of energy homeostasis. After suppression of ERα expression in this nucleus, female mice and rats developed a phenotype characteristic for metabolic syndrome and marked by obesity, hyperphagia, impaired tolerance to glucose, and reduced energy expenditure. This phenotype persisted despite normal ERα levels elsewhere in the brain. Although an increase in food intake preceded weight gain, our data suggest that a leading factor of obesity in this model is likely a decline in energy expenditure with all three major constituents being affected, including voluntary activity, basal metabolic rate, and diet-induced thermogenesis. Together, these findings indicate that ERα in the ventromedial nucleus of the hypothalamus neurons plays an essential role in the control of energy balance and the maintenance of normal body weight.