Nutrition is a highly modifiable risk factor for several types of cancer. The role of diets and dietary components in the prevention of breast cancer is well acknowledged, yet little is known about their biological effects in decreasing cancer susceptibility. The anti-tumor effects of soy protein isolate (SPI) and one of its bioactive component genistein (Gen) have been demonstrated in rat models of chemically induced mammary tumorigenesis by our laboratory and other groups. The present studies investigated the influence of developmental context on dietary protection against mammary tumorigenesis and revealed that limited exposure (in utero) to SPI and Gen positively influenced resistance to N-methy-N-nitrosourea (NMU)-induced mammary tumorigenesis later in life. We hypothesized that promotion of mammary gland differentiation status constitutes one mechanism by which dietary SPI and Gen confer tumor protection. To gain insights into the signaling pathways underlying enhanced mammary differentiation by these diets, we performed global gene expression analysis on mammary epithelial cells from young adult female rats and on whole mammary gland tissues of weanling female rats, which were lifetime exposed to SPI, Gen or control casein (CAS) diet, respectively. In mammary epithelial cells, we identified distinct SPI and Gen responsive genes. One of the signaling pathways that are specifically regulated by Gen is the Wnt/β-catenin signaling pathway. Mechanistically, we demonstrated that Gen increases/maintains cell adhesion E-cadherin protein levels to sequester β-catenin on the membrane, and thus, inhibit the proliferative effects of β-catenin. The induction of E-cadherin protein levels by Gen is mediated in part by the estrogen receptor β isoform. In prepubertal mammary tissues, SPI down-regulated the expression of lipogenesis related genes and influenced mammary adipocyte size. Dietary effects on mammary stroma and stroma-epithelial interactions were further investigated by using co-culture of mammary epithelial cells with conditioned medium from adipocytes in vitro. By treating differentiated 3T3-L1 adipocytes with Gen, we recapitulated the transcriptional regulation of mammary epithelium by SPI in vivo, and further demonstrated that Gen influences adipocyte secretion and local glucocorticoid signaling, thereby leading to enhancement of mammary differentiation. Results from these studies further our understanding of diet-mediated protective effects on the development of mammary tumorigenic lesions and may provide a molecular basis for prevention and treatment of breast cancer.