This study was to evaluate effects of milk replacer (MR) composition on: mass and composition of mammary parenchyma (PAR) and fat pad (MFP), growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis gene expression, and putative mammary epithelial stem cells. The hypothesis was that diet during the pre-weaning period alters the development, persistence, or activity of populations of putative mammary epithelial stem cells, possibly through involvement of GH/IGF-I axis molecules. Twenty-four newborn heifers were fed one of four MR diets: CON (20% CP, 21% fat MR fed at 441 g DM/d), HPLF (28% CP, 20% fat MR fed at 951 g DM/d), HPHF (27% CP, 28% fat MR fed at 951 g DM/d), and HPHF+ (27% CP, 28% fat MR fed at 1431 g DM/d). Animals were harvested on d 65 of life and mammary tissue was subjected to biochemical, molecular, and histological examination. By design, the effects of diet were evaluated at a common chronological age, but not necessarily at the same physiological age (body weight). Results from heifers reared on CON were compared to the average results from heifers reared on the other 3 diets. The second comparison evaluated the effect of increased fat in MR when protein content and intake were the same. The final comparison evaluated the effect of increased intake of a high-fat, high-protein MR. Neither diet composition nor nutrient intake in pre-weaned heifers affected PAR weight, PAR composition, GH/IGF-I axis gene expression, or putative mammary epithelial stem cell abundance when assessed at a common chronological age. Changes in MFP size and composition were observed, but no diet effect on GH/IGF-I axis gene expression in MFP was observed. This suggests nutrition is not critical for regulating the expression of local GH/IGF-I axis components or stem cell populations in the developing heifer mammary gland.