The objective of this study was to analyze the mammary gland transcriptome to determine how preweaning nutrient supply alters the molecular mechanisms that regulate preweaning mammary development. Holstein heifers were fed via milk replacer (MR) either an elevated level of nutrient intake (ELE; on average, 5.9 ± 0.2 Mcal of ME in 8.4 L of MR/d, n = 6) or a restricted amount of nutrients (RES; 2.8 ± 0.2 Mcal of ME in 4 L of MR/d, n = 5) for 54 d after birth, at which point they were slaughtered and samples of mammary parenchyma tissue were obtained. Parenchymal mRNA was analyzed, and the fold change (FC) of 18,111 genes (ELE relative to RES) was uploaded to Ingenuity Pathway Analysis (IPA) software (Qiagen Bioinformatics, Redwood City, CA) for transcriptomic analysis. Using a threshold of P < 0.05, IPA identified that the FC of 1,931 of 18,811 differentially expressed genes (DEG) could be used for the analysis. A total of 18 molecular and cellular functions were relevant to DEG arising from the treatments; the 5 functions most associated with DEG were cell death and survival, cellular movement, cellular development, cellular growth and proliferation, and lipid metabolism. Based on the directional FC of DEG, the mammary gland of ELE heifers was predicted to have increased epithelial-mesenchymal transition (Z = 2.685) and accumulation of lipid (Z = 2.322), whereas the synthesis of DNA (Z = −2.137), transactivation of RNA (Z = −2.254), expression of RNA (Z = −2.405), transcription (Z = −2.482), and transactivation (Z = −2.611) were all predicted to be decreased. Additionally, IPA predicted the activation status of 13 upstream regulators with direct influence on DEG as affected by ELE feeding that were ligand-dependent nuclear receptors (n = 2), enzymes (n = 1), or transcription regulators (n = 10). Of these, 6 were activated (Z > 2) and 7 were inhibited (Z < −2). In summary, feeding ELE preweaning altered the mammary transcriptome of Holstein heifers, affecting cell functions involved in the morphological and physiological development of the mammary gland.