Uncoupling protein 1 (UCP1) is a mitochondrial protein that is expressed in both brown and beige adipocytes. UCP1 uncouples the mitochondrial electron transport chain from ATP synthesis to produce heat via non-shivering thermogenesis. Due to their ability to dissipate energy as heat and ameliorate metabolic disorders, UCP1-expressing adipocytes are considered as a potential target for anti-obesity treatment. To monitor the expression of UCP1 in live mice in a non-invasive manner, we generated the Ucp1-iRFP720 knock-in (Ucp1-iRFP720 KI) mice, in which the gene encoding a near-infrared fluorescent protein iRFP720 is inserted into the Ucp1 gene locus. Using the heterozygous Ucp1-iRFP720 KI mice, we observed robust iRFP fluorescence in the interscapular region where brown adipose tissue is located. Moreover, the iRFP fluorescence was clearly observable in inguinal white adipose tissues in live mice administered with β3-adrenergic receptor agonist CL316,243. We also found that the homozygous Ucp1-iRFP720 KI mice, which are deficient in UCP1, displayed prominent iRFP fluorescence in the inguinal regions at the standard housing temperature. Consistent with this, the mice exhibited expanded populations of beige-like adipocytes in inguinal white adipose tissue, in which the Ucp1 promoter was dramatically activated. Thus, the Ucp1-iRFP720 KI mice provide a convenient model for non-invasive in vivo imaging of UCP1 expression in both brown and beige adipocytes in live mice.