The hypothalamic proopiomelanocortin (POMC) neurons act as first order sensors of systemic energy stores, providing signals that regulate caloric intake and energy expenditure. In experimental obesity, dietary saturated fatty acids affect POMC endopeptidases, resulting in the abnormal production of the neurotransmitters a-melanocyte stimulating hormone (a-MSH) and b-endorphin, thus impacting energy balance. The cAMP response element-binding protein (CREB) is one of the transcription factors that control the expression of POMC endopeptidases; however, it was previously unknown if dietary fats could affect CREB and consequently the expression of POMC endopeptidases. Here, we used single-cell RNA sequencing analysis, PCR, immunoblot, ELISA and immunofluorescence histological assays to determine the impact of a high-fat diet on the expression and function of hypothalamic CREB and its impact on the melanocortinergic system. The results indicate that CREB is expressed in arcuate nucleus POMC neurons and is activated shortly after the introduction of a high-fat diet. The inhibition of hypothalamic CREB using a short-harpin RNA lentiviral vector resulted in increased diet-induced body mass gain and reduced energy expenditure. This was accompanied by reduced expression of the POMC endopeptidases Pcsk2, Prcp and Cpe and by the loss of the high-fat diet-induced effect of a-MSH inhibition. This study provides the first evidence for the involvement of CREB in the abnormal regulation of the hypothalamic POMC endopeptidase system in experimental obesity