The present study was designed to investigate the role of the medial preoptic nucleus (MPO) as a site of the thermogenic and metabolic effects of the αMSH analog Melanotan II (MTII). We also assessed the involvement of the dorsomedial hypothalamic nucleus (DMH) by investigating the effects of the MPO infusion of MTII in rats with DMH lesions produced by kainic acid. Infusion of MTII in the MPO led to increases in interscapular BAT (iBAT) temperature and iBAT uptake of ¹⁴C-bromopalmitate. Both increases were blocked by DMH lesions. iBAT temperature increase (area under curve) and ¹⁴C-bromopalmitate uptake emerged as two correlated variables (r = 0.63, P < 0.001). DMH lesions also blocked MTII-induced expression of mRNAs coding for proteins involved in (i) thermogenesis [type II iodothyronine deiodinase (Dio2) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α)], (ii) lipolysis [hormone sensitive lipase (Hsl)] and (iii) lipogenesis [diacylglycerol O-acyltransferase 2 (Dgat2), fatty acid synthase (Fas)], in iBAT of rats killed one hour after MPO infusion of MTII. MTII also stimulated expression of genes in iWAT but only in rats with DMH lesions. These genes included glucose transporter member 4 (Glut4), glycerol-3-phosphate acyltransferase 3 (Gpat3), Dgat1, Dgat2, triglyceride lipase (Atgl), Hsl and carnitine palmitoyltransferase 1β (Cpt1β). Altogether, the present results reveal the MPO as a site of the thermogenic and metabolic actions of MTII. They also contribute to establish the MPO-DMH duet as a significant target for melanocortins to modulate energy homeostasis.