Vitamin D3 supplementation is accepted as a therapeutic in many common estrogen-responsive cancers. In breast cancer, a metabolite of vitamin D3, 24R,2As5(OH)2D3, has an anti-tumorigenic effect depending on the expression levels of estrogen receptor alpha(ERα) isoforms, specifically ERα66. In ER+ cells, which express both the canonical estrogen receptor ERα66 and the ER splice variant, ERα36, 24R,25(OH)2D3 inhibits proliferation, tumorigenesis, and production of metastatic markers. In ER- cells lacking ERα66 but expressing ERα36, it has the opposite effect. 24R,25(OH)2D3 acts via a membrane-associated mechanism involving PLC-dependent activation of PKCα, leading to activation of MAPK. Membrane-mediated effects of 17β-estradiol also result in downstream activation of MAPK, suggesting interaction between the two mechanisms. Mouse studies confirm the tumorigenic effects of 24R,25(OH)2D3. Treatment with 24R,25(OH)2D3 increased tumor number and volume when implanted with cells lacking ERα66, and reduced when implanted with ERα66+ cells. Treatment of mice with 24R,25(OH)2D3 increased breast cancer tumor number and volume in bone. Breast cancer cells produce their own 24,25(OH)2D3 locally, indicating that the tumor can regulate itself via paracrine/autocrine signaling. To test if this effect of 24R,25(OH)2D3 is unique to breast cancer cells, we examined it's role in estrogen-sensitive laryngeal cancer. ERα66+UM-SCC-12 cells[UM12] and ERα66-UM-SCC-11A cells[UM11A] express CYP24A1 and CYP27B1. When treated with 25(OH)D3, UM11A cells produced higher amounts of 24,25(OH)2D3 compared to UM12 cells, at physiologically relevant levels observed in breast cancer. Both laryngeal cell lines produced similar levels of 1,25(OH)2D3. In contrast to the breast cancer cells, 24R,25(OH)2D3 increased proliferation and inhibited apoptosis in ERα66+ UM12 cells in vitro, and had the opposite effect in ERα66- UM11A cells. 24R,25(OH)2D3 acts via a membrane-associated mechanism involving PLC-dependent activation of PKCα in these cells. Mice with UM11A tumors given 24R,25(OH)2D3 had reduced tumor burden and increased survival compared to vehicle-treated controls. Mice with UM12 tumors given 24R,25(OH)2D3 showed increased tumor burden and reduced survival compared to controls. These observations support our earlier findings examining the effects of 24R,25(OH)2D3 in breast cancer, indicating that the vitamin D metabolite acts via a mechanism involving the presence of ERα66 and works via autocrine/paracrine signaling pathways, although the specific effects of 24R,25(OH)2D3 vary with cancer type.