Abstract 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), the most active vitamin D metabolite, regulates proliferation, survival, and differentiation in many cell types. 1,25(OH)2D3 and several less calcemic analogs are in clinical trials against various neoplasias. We studied the effects of 1,25(OH)2D3 on a panel of human breast cancer cells, which show similar vitamin D receptor (VDR) content but variable transcriptional and anti-proliferative responsiveness. In MDA-MB-453 cells, one of the responsive lines, 1,25(OH)2D3 increased cell and nuclear size and induced a change from a rounded to a flattened morphology. By phase contrast, laser confocal and electron microscopy, we found that 1,25(OH)2D3 changed the cytoarchitecture of actin filaments and microtubules and nuclear shape, induced filopodia and lamellipodia, and promoted cell-to-cell contacts via large cytoplasmic extensions. However, although claudin-7 and occludin content in the cells increased upon exposure to 1,25(OH)2D3, these proteins were not located at the plasma membrane probably due to the absence of E-cadherin expression. Additionally, 1,25(OH)2D3 induced the accumulation of αv-integrin, β5-integrin, focal adhesion kinase (FAK), and paxillin in focal adhesion plaques, concomitant with the increased phosphorylation of the FAK. 1,25(OH)2D3 enhanced MDA-MB-453 and MDA-MB-468 cell adhesion to plastic but decreased adhesion to laminin. The expression of the mesenchymal marker N-cadherin and of the myoepithelial marker P-cadherin was down-regulated by 1,25(OH)2D3 in several breast cancer cell lines. Other myoepithelial proteins such as α6-integrin, β4-integrin, and smooth muscle α-actin (SMA) were also repressed by 1,25(OH)2D3 in MDA-MB-453 and MDA-MB-468 cells. Accordingly, mice lacking VDR (Vdr−/−) showed abnormally high levels of SMA and P-cadherin in their mammary gland. These findings show that 1,25(OH)2D3 profoundly affects the phenotype of breast cancer cells, and suggest that it reverts the myoepithelial features associated with more aggressive forms and poor prognosis in human breast cancer.