Both ovarian and pituitary hormones are required for the pubertal development of the mouse mammary gland. Estradiol directs ductal elongation and branching, while progesterone leads to tertiary branching and alveolar development. The purpose of this investigation was to identify estrogen-responsive genes associated with pubertal ductal growth in the mouse mammary gland in the absence of other ovarian hormones and at different stages of development. We hypothesized that the estrogen-induced genes and their associated functions at early stages of ductal elongation would be distinct from those induced after significant ductal elongation had occurred. Therefore, ovariectomized prepubertal mice were exposed to 17β-estradiol from two to twenty-eight days, and mammary gland global gene expression analyzed by microarray analysis at various times during this period. We found that: a) gene expression changes in our estrogen-only model mimic those changes that occur in normal pubertal development in intact mice, and b) both distinct and overlapping gene profiles were observed at varying extents of ductal elongation, and c) cell proliferation, the immune response, and metabolism/catabolism were the most common functional categories associated with mammary ductal growth. Particularly striking was the novel observation that genes active during carbohydrate metabolism were rapidly and robustly decreased in response to estradiol. Lastly, we identified mammary estradiol-responsive genes that are also co-expressed with Estrogen Receptor α in human breast cancer. In conclusion, our genomic data support the physiological observation that estradiol is one of the primary hormonal signals driving ductal elongation during pubertal mammary development.