Objectives were to study how pre-weaning nutrition and estradiol alter the mammary transcriptome of Holstein calves using RNA-sequencing. Twenty-four Holstein calves were assigned to 2 experimental milk replacers (MR, n = 12/group): control (CON, 20.9% CP, 19.8% fat) MR fed at 0.44 kg/day, or enhanced MR (HIG, 28.9% CP, 26.2% fat) fed at 1.08 kg/day. 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. MR was fed for 8 weeks. At the end of wk 8, calves were given an estradiol (E2) implant or placebo. Treatment groups were (n = 6/group): CON plus placebo (R), CON plus E2 (R-E2), HIG plus placebo (EH), and HIG plus E2 (EH-E2). Two weeks post-E2 all calves were sacrificed and mammary parenchyma (PAR) and fat pad (MFP) removed and snap-frozen in liquid-N until RNA extraction. Twenty-two (10 upregulated, 12 downregulated) and 351 (69 upregulated, 282 downregulated) differentially expressed genes (DEG, adjusted p-value <0.05, fold change (FC) > |1.5|) were detected in the MFP in response to HIG and E2, respectively. A total of 369 (210 upregulated, 159 downregulated) and 427 (103 upregulated, 324 downregulated) DEG were identified in the PAR in response to HIG and E2, respectively. Bioinformatics analysis revealed that in the MFP, the most-impacted metabolic pathways by HIG included ‘Metabolism of Cofactors and Vitamins’, ‘Amino Acid Metabolism’, and ‘Carbohydrate Metabolism’. Among the most-impacted pathways by E2 were ‘Lipid Metabolism’, ‘Amino Acid Metabolism’, and ‘Glycan Biosynthesis and Metabolism’, with most being downregulated. Upstream transcription regulator analysis of DEG affected by HIG and E2 using Ingenuity Pathway Analysis identified 17 and 4 transcription regulators (TR) in MFP, while 12 and 7 TR in PAR, respectively. Several downstream upregulated and downregulated target genes are linked with these TR, underscoring the role of molecular networks on tissue development in response to HIG and E2. These results provide an integrated understanding of the molecular basis of how pre-weaning nutrition and post-weaning exogenous estrogen regulate mammary development.