The regulation of energy homeostasis is complex, however key physiological systems have been identified. The melanocortin system has been demonstrated to be a master regulator of energy homeostasis. Within the melanocortin system are the melanocortin-3 or -4 receptors (MC3R & MC4R). Global MC4R deletion results in profound hyperphagia and obesity, even on regular chow. In stark contrast, global deletion of MC3R doesn’t change food intake on normal chow. Furthermore, the MC3RKO mouse gains more weight than WT mice under some conditions (e.g. high fat diet or ovariectomy) but loses more weight than WT mice under others (diet, food restriction, or cachexia). However, how the MC3RKO regulates diet induced obesity remained understudied. Using the MC3RKO mouse, I found that when challenged with a high fat diet chronically, they continuously gain weight, become hyperphagic, and are sensitive to leptin. To further determine if loss of MC3R in the ventromedial hypothalamic nucleus (VMH) or arcuate nucleus (ARH) recapitulated the enhanced diet-induced obesity phenotype, I deleted Mc3r from SF1 neurons in the VMH and from AgRP neurons in the ARH using an Mc3r flox mouse. Interestingly, I found that fat mass was slightly improved in a sex specific manner in the SF1-Cre; Mc3r FL/FL, as well as glucose handling. In the AgRP-Cre; Mc3r FL/FL mice, I also found that glucose handling was improved in a sex specific manner. Next in collaboration with the Schwendemann lab, we found that delivery of the MC4R agonist therapeutic, setmelanotide, when packaged in a poly-lactic-glycolic microsphere formulation improved body weight up to 1 month in diet-induced obese mice. To investigate the transcriptional landscape of MC3R neurons, I performed single nucleus RNAseq using the Sun1-GFP system. We identified known clusters expressing Mc3r, but also found several clusters that express Mc3r, which have not been reported. Taken together, my dissertation demonstrates that the loss of Mc3r regulates fat, glucose, and leptin in a dynamic manner.