To study the effect of estrogen on mammary gland development in young heifers, mammary parenchymal tissue (PAR) was collected at weaning (8 wk) from 5 Holstein heifer calves reared on an accelerated milk replacer (1.13 kg/d, 28% crude protein, 25% fat), and incubated for 6h at 3 estrogen concentrations: 0, 10, and 100 pg/mL. RNA was extracted and sequenced on the Illumina HiSeq 4000 system, generating an average of 27 million reads/sample of which 94% where uniquely mapped, with 81% of gene-assigned reads. A linear model with estrogen dose as fixed and animal as random effect was fitted. Differentially expressed genes (DEG) were declared at FDR ≤0.2 and raw p-value ≤0.01. The Dynamic Impact Approach was used for pathway analyses to determine effect on biological pathways. DEG (791) were detected only for the comparison of 100 vs 0 pg/mL, with 435 and 356 up and downregulated genes, respectively. As expected, the estrogen signaling pathway was upregulated when explants were incubated with 100 pg/mL of estrogen. Among the top 15 impacted metabolic pathways, there was an overall upregulation of fatty acid metabolism and a downregulation of amino acid biosynthesis. These were driven by an upregulation of steroid biosynthesis, and lysine and β-Alanine catabolism, that together with the upregulation of DNA replication, cell cycle, AMPK and Wnt signaling pathways (among the top 15 impacted non-metabolic pathways) suggest a higher proliferating state of the cells. At a metabolic level, proliferation was supported by upregulation of purine metabolism and mucin type O-glycan biosynthesis. Furthermore, explants incubated with 100 pg/mL of estrogen had a downregulation of immunometabolic pathways (e.g., cytokine-cytokine receptor interaction, and chemokine and adipocytokine signaling pathways). Overall, results suggest a stimulation of growth and proliferation of PAR by exposure to estrogen at early stages of life in heifer calves fed an accelerated milk replacer.