Abstract The biologically active form of vitamin D (1,25(OH)2D) regulates proliferation, differentiation, and apoptosis in diverse cell types. We have previously identified anti-proliferative activities of 1,25(OH)2D in human breast tissue, as well as 1,25(OH)2D target gene expression consistent with reports that elevated vitamin D levels may protect against cancer. In mouse studies, vitamin D signaling modulates normal mammary gland development, including ductal outgrowth and branching, and protects against tumorigenesis. Degradation of 1,25(OH)2D is initiated by the enzyme Cyp24a1 in target tissues, providing critical local control of 1,25(OH)2D bioactivity. In vitro, blockade of Cyp24a1 activity potentiates the anti-proliferative effects of 1,25(OH)2D. However, the extent to which endogenous Cyp24a1 activity within the mammary epithelium regulates local 1,25(OH)2D levels to modulate normal mammary gland development, with possible implications for cancer, has not been investigated. We generated a novel mouse model with conditional knockout of the Cyp24a1 gene specifically in the mammary epithelium (MMTV-Cre x Cyp24a1lox/lox). Ablation of Cyp24a1 activity in the mammary epithelium does not alter either gland or body weight at 4, 6 or 10 weeks of age. Preliminary analyses of mammary gland whole mounts indicate that virgin knockout mice form fewer terminal end buds compared to glands from wild-type littermates at 4 and 6 weeks of age (P<0.05). Moreover, the width of the ducts proximal to the central lymph node of knockout mice was less than that of wild-type mice at 4 and 10 weeks of age (P<0.05 and P<0.01, respectively). In addition, the number of secondary and tertiary branching points is reduced in mammary glands from knockout mice at 6 weeks of age (P<0.05 and P<0.01, respectively). In summary, our findings suggest that Cyp24a1 activity within epithelial cells plays a crucial role to modulate postnatal mammary gland development, presumably by limiting the local accumulation of 1,25(OH)2D. Citation Format: Sheng L, Turner AG, Tarulli GA, Barratt K, Kremer R, Morris HA, Callen DF, Anderson PH. Conditional inactivation of the 25-hydroxyvitamin D-24-hydroxylase (Cyp24a1) in the mouse mammary epithelium alters mammary gland development [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-05-02.