Vitamin D3 (vitD3) and its active metabolite, calcitriol (1,25-(OH)2D3), affect multiple tissue types by interacting with the vitamin D receptor (VDR). Although vitD3 deficiency has been correlated with increased incidence of breast cancer and less favorable outcomes, randomized clinical trials have yet to provide conclusive evidence on the efficacy of vitD3 in preventing or treating breast cancer. Additionally, experimental studies are needed to assess the biological plausibility of these outcomes. The mammary gland of VDR KO mice shows a florid phenotype revealing alterations of developmental processes that are largely regulated by mammotropic hormones. However, most research conducted on vitD3's effects used 2D cell cultures and supra-physiological doses of vitD3, conditions that spare the microenvironment in which morphogenesis takes place. We investigated the role of vitD3 in mammary epithelial morphogenesis using two 3D culture models. VitD3 interfered with estrogen's actions on T47D human breast cancer cells in 3D differently at different doses, and recapitulated what is observed in vivo. Also, vitD3 can act autonomously and affected the organization of estrogen-insensitive MCF10A cells in 3D collagen matrix by influencing collagen fiber organization. Thus, vitD3 modulates mammary tissue organization independent of its effects on cell proliferation.