Summary Thermogenic brown and beige adipocytes convert chemical energy to heat by metabolizing glucose and lipids. Serotonin (5-HT) neurons in the central nervous system are essential for thermoregulation and accordingly may control metabolic activity of thermogenic fat. To test this, we generated mice in which the human diphtheria toxin receptor was selectively expressed in central 5-HT neurons. Treatment with diphtheria toxin eliminated 5-HT neurons and caused loss of thermoregulation, brown adipose tissue (BAT) steatosis, and a >50% decrease in uncoupling protein 1 (Ucp1) expression in BAT and inguinal white adipose tissue (WAT). In parallel, blood glucose increased 3.5-fold, free fatty acids 13.4-fold and triglycerides 6.5-fold. Similar BAT and beige fat defects occurred in Lmx1bf/f/p mice, in which 5-HT neurons fail to develop in utero. We conclude 5-HT neurons play a major role in regulating glucose and lipid homeostasis, in part through recruitment and metabolic activation of brown and beige adipocytes.