1,25(OH)2D3 antiproliferative properties are widely known. However, the molecular bases of these properties are only partially elucidated. Since 1,25(OH)2D3 effectively arrests growth in many tumors and hyperplastic tissues whose growth is driven by co-expression of EGFR and its ligand TGF-α, it was hypothesized that 1,25(OH)2D3 could affect the TGF-α/EGFR-autocrine growth loop. This study examined 1,25(OH)2D3 regulation of EGFR-growth signals, using human epidermoid A431 cells, in which the overexpression of EGFR and TGF-α constitute the major autocrine mitogenic signal. 1,25(OH)2D3 inhibited autocrine and EGF-induced A431 cell proliferation. Furthermore, 1,25(OH)2D3 changed the cellular localization of both TGF-α and EGFR and inhibited ligand-dependent phosphorylation of EGFR and ERK1/2. In addition, 1,25(OH)2D3 impaired autocrine and EGF-induced nuclear translocation of activated EGFR and, consequently, its binding to AT-rich DNA sequences and transcriptional activation of the cyclin D1 promoter. These results demonstrate that 1,25(OH)2D3 alters EGFR membrane trafficking and down-regulates EGFR growth signaling.