Objectives were to study how pre-weaning nutrition alters the mammary transcriptome of Holstein calves using RNA-sequencing. The control (CON) group was fed 0.44 kg/d of milk replacer (MR, 20.9% CP, 19.8% fat), while the higher-plane of nutrition group (HIG, 28.9% CP, 26.2% fat) was fed 1.08 kg/d of MR. Starter feed was introduced at the end of wk 4 and MR was reduced in both treatments to 50% at wk 8 to induce weaning. Calves (n = 6 per diet) were sacrificed upon weaning (wk 8). Parenchyma (PAR) and mammary fat pad (MFP) tissue were removed and snap-frozen in liquid-N until RNA extraction. A total of 167 differentially expressed genes (DEG) (adjusted p-value <0.05, fold change > |1.5|) in the MFP and 487 DEG in the PAR were detected in response to HIG. Of those, 108 were upregulated and 59 were downregulated in the MFP and 319 were upregulated and 168 were downregulated in the PAR. The Dynamic Impact Approach with the Kyoto Encyclopedia of Genes and Genomes pathway database was used for bioinformatics analysis of DEG. In the MFP, the most-impacted pathways by HIG included ‘Glycan Biosynthesis and Metabolism’, ‘Metabolism of Cofactors and Vitamins’, ‘Lipid Metabolism’, and ‘Carbohydrate Metabolism’. In PAR, most-impacted pathways included ‘Lipid Metabolism’, ‘Amino Acid Metabolism’, ‘Metabolism of Cofactors and Vitamins’, ‘Energy Metabolism’, and ‘Glycan Biosynthesis and Metabolism’. Upstream transcription regulator analysis of DEG affected by HIG (fold change > |1.5| versus CON) using Ingenuity Pathway Analysis identified 4 transcription regulators (TR) in MFP (FOS, EGR1, ZFP36, and EGR2) and 16 in PAR (DLX4, PAX1, EGR2, FOXM1, E2F7, E2F2, E2F3, CCNE1, BRCA1, KLF4, TP73, KLF15, FOSB, PER2, MYOCD, and FOS). Several downstream upregulated and downregulated target genes were associated with these TR, underscoring the role of molecular networks on tissue development in response to HIG. These results allow a more comprehensive understanding of the molecular mechanisms whereby pre-weaning nutrition regulates mammary development. The relevance of these molecular alterations to future milk production performance remain to be determined.