Within the central neural circuitry for thermoregulation, the balance between excitatory and inhibitory inputs to the dorsomedial hypothalamus (DMH) determines the level of activation of brown adipose tissue (BAT) thermogenesis. We employed neuroanatomical and in vivo electrophysiological techniques to identify a source of excitation to thermogenesis-promoting neurons in the DMH that is required for cold defense and fever. Inhibition of median preoptic area (MnPO) neurons blocked the BAT thermogenic responses during both PGE2-induced fever and cold exposure. Disinhibition or direct activation of MnPO neurons induced a BAT thermogenic response in warm rats. Blockade of ionotropic glutamate receptors in the DMH, or brain transection rostral to DMH, blocked cold-evoked or NMDA in MnPO-evoked BAT thermogenesis. RNAscope technique identified a glutamatergic population of MnPO neurons that projects to the DMH and expresses c-Fos following cold exposure. These discoveries relative to the glutamatergic drive to BAT sympathoexcitatory neurons in DMH augment our understanding of the central thermoregulatory circuitry in non-torpid mammals. Our data will contribute to the development of novel therapeutic approaches to induce therapeutic hypothermia for treating drug-resistant fever, and for improving glucose and energy homeostasis.