Stimulation of brown adipose tissue (BAT) thermogenesis in humans has emerged as an attractive target to improve metabolic health. Pharmacological stimulations targeting the β₃-adrenergic receptor (β₃-AR), the adrenergic receptor believed to mediate BAT thermogenesis, have historically performed poorly in human clinical trials. Here we report that, in contrast to rodents, human BAT thermogenesis is not mediated by the stimulation of β₃-AR. Oral administration of the β₃-AR agonist mirabegron only elicited increases in BAT thermogenesis when ingested at the maximal allowable dose. This led to off-target binding to β₁-AR and β₂-AR, thereby increasing cardiovascular responses and white adipose tissue lipolysis, respectively. ADRB2 was co-expressed with UCP1 in human brown adipocytes. Pharmacological stimulation and inhibition of the β₂-AR as well as knockdown of ADRB1, ADRB2, or ADRB3 in human brown adipocytes all confirmed that BAT lipolysis and thermogenesis occur through β₂-AR signaling in humans (ClinicalTrials.gov NCT02811289).