F-FDG PET imaging is routinely used to investigate brown adipose tissue (BAT) thermogenesis, which requires mitochondrial uncoupling protein 1 (UCP1). It remains uncertain, however, whether BAT F-FDG uptake is a reliable surrogate measure of UCP1-mediated heat production. UCP1 knockout (KO) and wild-type (WT) mice housed at thermoneutrality were treated with the selective β3 adrenergic receptor agonist CL 316, 243 and underwent metabolic cage, infrared thermal imaging and F-FDG PET/MRI experiments. Primary brown adipocytes were additionally examined for their bioenergetics by extracellular flux analysis as well as their uptake of 2-deoxy-H-glucose. In response to CL 316, 243 treatments, oxygen consumption, and BAT thermogenesis were diminished in UCP1 KO mice, but BAT F-FDG uptake was fully retained. Isolated UCP1 KO brown adipocytes exhibited defective induction of uncoupled respiration whereas their glycolytic flux and 2-deoxy-H-glucose uptake rates were largely unaffected. Adrenergic stimulation can increase BAT F-FDG uptake independently of UCP1 thermogenic function.