Epigenetic regulation of neuropeptide genes associated with central appetite control plays an important part in the development of nutritional programming. While proopiomelanocortin (POMC) is critical in appetite control, the molecular mechanism of methylation-related regulation of POMC remains unclear. Based on the report that the proximal specificity protein 1 (Sp1) binding site in POMC promoter is crucial for the leptin-mediated activation of POMC, the methylation of this site was investigated in this study in both cultured cells and postnatal mice reared by the dams with dietary supplementation of conjugated linoleic acids (CLAs). The change of milk composition made the offspring undergo the increase of food intake, suppression of POMC, attenuation of Sp1-promoter interaction, and the hypermethylation of cytosine guanine (CpG) dinucleotides at -100 and -103 within the Sp1 binding site of POMC promoter, which may be associated with the decrease of hypothalamic Sp1 and/or plasma S-adenosylhomocystein. In cultured cells, the methylation of the -100 CpG dinucleotides of the POMC promoter blocked both the formation of Sp1-promoter complex and the leptin-induced activation of POMC. In addition, a catch-up growth and adult metabolic changes like adult hyperglycemia and insulin resistance were observed in these postnatal pups, suggesting that this CLA-mediated hypermethylation may contribute, at least in part, to the metabolic disorders.