Early developmental events influence the fine tuning of later susceptibility to adult diseases. Diet is a determinant of breast cancer risk, and our previous studies showed that diet-mediated changes in transcriptional programs promote early mammary gland differentiation. Although consumption of fruits is considered to elicit multiple health benefits, little is known on whether associated bioactive components modify the early differentiation program in developing mammary glands. Here, we evaluated the hypothesis that early exposure (in utero and lactational) to blueberry through maternal diet enhances mammary epithelial differentiation in female offspring. Pregnant Sprague-Dawley rats beginning at gestation day 4 were fed American Institute of Nutrition-based diets containing casein and whole blueberry powders added to casein at 2.5%, 5.0%, and 10% weight/weight. Female pups at weaning were evaluated for growth and mammary tissue parameters. Blueberry at 5% dose increased body and adipose fat weights, relative to the other diets. Mammary branch density and terminal end bud size were highest for the 5% blueberry group, whereas terminal end bud numbers were not affected by all diets. Mammary ductal epithelial cells of the 5% blueberry group had lower nuclear phosphorylated histone 3 and higher nuclear tumor suppressor phosphatase and tensin homolog deleted in chromosome 10 (PTEN) levels than the casein group. Although sera of both diet groups had similar antioxidant capacity, 5% blueberry sera elicited higher nuclear PTEN accumulation in human MCF-10A mammary epithelial cells. Our studies identify developing mammary glands as early targets of blueberry-associated bioactive components, possibly through systemic effects on epithelial PTEN signaling.