Vitamin D3 (vitD3) has been implicated in various cellular functions affecting multiple tissue types. Epidemiological and laboratory studies suggest that vitD3 may be effective as a preventive or therapeutic option for breast cancer. However, randomized clinical trials have yet to confirm these suggestions. Breast neoplasias can arise from developmental alterations; based on this evidence, we seek to understand vitD3's role in normal breast development, particularly its role in epithelial morphogenetic processes such as ductal elongation, branching, and alveolar formation. These processes require extensive changes in the extracellular microenvironment, such as collagen fiber organization, and are largely influenced by hormones. Here, we build upon our past work to shed light on calcitriol's effects on collagen fiber organization by breast epithelial cells, and how such effects are modulated by extracellular matrix composition. We embedded MCF10A normal human breast epithelial cells in two different matrices-collagen type I and collagen type I + 10% Matrigel; treatment with calcitriol resulted in flatter epithelial structures. Next, using two-photon microscopy, we examined changes in collagen fiber organization and corresponding changes in epithelial structures. Applying a novel three-dimensional (3D) image analysis method, we show that increasing doses of calcitriol result in denser collagen fiber bundles in the localized area surrounding the epithelial structures, and that these bundles are aligned in a more parallel direction to epithelial structures when exposed to the highest vitD3 dose. Changed patterns in fiber organization may explain the flattening of epithelial structures; in turn, changes in biophysical forces in the matrix abutting these structures may be responsible for changes in the referred patterns. Addition of 10% Matrigel dampened the effects of calcitriol on both epithelial morphogenesis and fiber organization. Overall, we report novel functions of calcitriol in the breast epithelium and add to the growing body of evidence documenting how hormones affect biophysical processes. Impact statement In this study, we report novel functions of calcitriol in the breast epithelium and use a novel quantitative metric to parse the effects of calcitriol on collagen fiber organization that cannot be detected through conventional histological procedures. Despite the large body of literature on vitamin D3 (vitD3) and calcitriol's effects on cellular functions across tissue types, little is known about how they affect collagen fiber organization, an early critical step for breast epithelial development. This work provides further evidence that hormones affect morphogenesis by means of biophysical forces, with implications for a comprehensive view on vitD3's effects in breast development and neoplasia.