The circadian clock plays a critical role in coordinating energy metabolism across tissues, including brown adipose tissue (BAT), a major site of nonshivering thermogenesis. This study aimed to elucidate the cell-autonomous role of the peripheral circadian clock in brown adipocyte thermogenesis using an in vitro model independent of extrinsic cues. Primary brown adipocytes were differentiated from the stromal vascular fraction of interscapular BAT isolated from C57BL/6J mice. An in vitro model of BAT clock disruption was established by siRNA-mediated knockdown of the core clock gene Bmal1. Thermogenic function was assessed via measurement of oxygen consumption rate (OCR) using an extracellular flux analyzer. To further assess the thermogenic process, protein expression levels of lipolytic enzymes and mitochondrial oxidative phosphorylation (OXPHOS) complexes were analyzed by Western blotting. Bmal1 knockdown markedly reduced both basal and β-adrenergic-stimulated OCR, indicating impaired thermogenic function, despite comparable cellular differentiation, preserved β-adrenergic responsiveness, and elevated uncoupling protein 1 (UCP1) expression. Notably, Bmal1-deficient cells exhibited decreased protein expression of key lipolytic enzymes, adipose triglyceride lipase and hormone-sensitive lipase, as well as multiple mitochondrial OXPHOS subunits, suggesting decreased free fatty acid supply and reduced mitochondrial ability to generate the proton gradient required for UCP1-mediated thermogenesis. The peripheral circadian clock in brown adipocytes supports thermogenic function by regulating lipid mobilization and mitochondrial oxidative function; thus its disruption may lead to decreased energy expenditure and increased susceptibility to metabolic disorders.