The objective of this study was to examine the effects of a diet with high level of energy and protein, combined with bST injections, on leptin and leptin-receptor (Ob-Rb) gene expression profile in the mammary parenchyma of dairy heifers. Mammary parenchyma samples from 32 Holstein heifers, randomly assigned to one of four treatments (n=8), were utilized: high or low energy and protein diet, with or without bST injection. The experiment was designed in randomized blocks and arranged in a 2 x 2 factorial arrangement. Total RNA was extracted from tissue samples and gene expression profile was tested in qRT-PCR, against glucuronidase beta as a control, using the 2(-Delta Delta Ct) method. Animals receiving the diet with high level of energy and protein had higher leptin mRNA expression, with an increase of 56%, and lower leptin receptor Ob-Rb mRNA expression, with a decrease of 18%. On the other hand, bST injection decreased leptin mRNA and receptor Ob-Rb mRNA expression in 74% and 23%, respectively. There was no interaction between diet and bST injection. The increase in leptin expression can explain, at least in part, the detrimental effects caused by diets rich in energy and protein, offered during the prepubertal period, in future milk production of dairy heifers.