The Vitamin D3 Receptor (VDR) is present in all microenvironments of the breast, yet is hypothesized to signal through the epithelium to regulate hormone induced growth and differentiation. However, the influence or contribution of the other microenvironments within the breast that express VDR, like the breast adipose tissue, have yet to be investigated. We hypothesized that the breast adipocytes express the signaling components necessary to participate in vitamin D3 synthesis and signaling via VDR, modulating ductal epithelial cell growth and differentiation. We utilized human primary breast adipocytes and VDR wild type (WT) and knockout (KO) mice to address whether breast adipocytes participate in vitamin D3-induced growth regulation of the ductal epithelium. We report in this study that breast primary adipocytes express VDR, CYP27B1 (1α-hydroxylase, 1α-OHase), the enzyme that generates the biologically active VDR ligand, 1α,25-dihydroxyvitamin D3 (1,25D3), and CYP24 (24- hydroxylase, 24-OHase), a VDR-1,25D3 induced target gene. Furthermore, the breast adipocytes participate in bioactivating 25-hydroxyvitamin D3 (25D3) to the active ligand, 1,25D3, and secreting it to the surrounding microenvironment. In support of this concept, we report that purified mammary ductal epithelial fragments (organoids) from VDR KO mice, co-cultured with WT breast adipocytes, were growth inhibited upon treatment with 25D3 or 1,25D3 compared to vehicle alone. Collectively, these results demonstrate that breast adipocytes bioactivate 25D3 to 1,25D3, signal via VDR within the adipocytes, and release an inhibitory factor that regulates ductal epithelial cell growth, suggesting that breast adipose tissue contributes to vitamin D3-induced growth regulation of ductal epithelium.