Abstract We have reported that chronic treatment with β3-adrenoceptor agonists reduces body fat content and induces the expression of mitochondrial thermogenic uncoupling protein 1 (UCP1) in adipose tissue in the dog. To evaluate the role of UCP1 in the anti-obesity effect of the agonists, we isolated adipocytes from subcutaneous fat pad of beagles before and after a 2-week treatment with AJ-9677, a specific β3-adrenoceptor agonist, and examined their thermogenic activity in vitro. Histological and protein analysis revealed that adipose tissues before the treatment were composed of unilocular cells filled with a single large droplet, while the tissues after the treatment contained many smaller and some multilocular adipocytes expressing UCP1 and abundant mitochondrial proteins. Before the treatment, oxygen consumption rate was very low and did not change even when the cells were stimulated by AJ-9677. Two-week AJ-9677 treatment increased basal oxygen consumption rate by 7-fold, and produced a clear responsiveness to AJ-9677 stimulation. Thus, chronic treatment with AJ-9677 induced UCP1 in adipocytes, where oxygen consumption increased in response to AJ-9677 stimulation. It was suggested that UCP1-dependent energy expenditure in adipose tissue contributes to the anti-obesity effect of β3-adrenoceptor agonist in dogs.