An enhanced pre-weaning diet improves development of the immature, bovine mammary gland. Alterations in the population of myoepithelial cells (MYEC) can also affect mammary development. Our objective was to determine the impact of a higher plane of nutrition (HPN) on the MYEC population within the mammary gland of dairy calves. Thirty-six Holstein heifer calves were reared on (1) a control milk replacer (MR) fed at 454 g powder/d (R; 20% CP and 20% fat) or (2) an enhanced MR fed at 1,135 g powder/d (EH; 28% CP and 25% fat). At weaning (wk 8), a subset of calves (n = 6/diet) were harvested. Post-weaning, half of the remaining calves from each diet were supplemented with exogenous estradiol (E₂) to determine if a 2 week mammogenic stimulation further affected the MYEX population in response to diet consumed. At harvest, the mammary parenchyma (PAR) was removed and fixed. MYEC nuclei were identified by immune-staining for p63 in sections from lower, middle and distal portions of PAR. Images were analyzed using CellProfiler to measure nuclear characteristics of the MYEC population within each image. Comparisons between treatments were determined using PROC GLIMMIX in SAS. Nuclear area of MYEC was greater for calves fed EH compared with R (675 vs. 620 pixels; P < 0.05). Additionally, calves fed EH had a greater number of neighboring MYEC surrounding a given cell compared with calves fed R (5.2 vs. 4.9 neighbors; P < 0.01) and had a greater nuclear perimeter (105 vs. 99 pixels; P < 0.05). Nuclear compactness and eccentricity (degree of elongation) were not affected by dietary treatment (P = 0.99 and 0.84, respectively). Additionally, diet did not affect the distance between a given MYEC and its closest neighboring cell (P = 0.28). Finally, E₂ supplementation did further impact the MYEX population. These results suggest that diet-induced changes in prepubertal mammary growth are linked to alterations in MYEC population.