Active vitamin D (1,25(OH)2D) has been shown to regulate numerous cell processes in mammary cells. Degradation of 1,25(OH)2D is initiated by the mitochondrial enzyme 25-hydroxyvitamin D 24-hydroxylase (CYP24A1), and provides local control of 1,25(OH)2D bioactivity. CYP24A1 is coamplified with a known oncogene in human breast cancer tissue, and thus CYP24A1 may be an important player in contributing to the dysregulation of cell growth through lowering local cellular 1,25(OH)2D production. To assess the role of CYP24A1 activity in normal mammary development, we used mammary epithelial-specific Cyp24a1 knockout mice to demonstrate reduced terminal end bud number, ductal outgrowth and branching during puberty and alveologenesis during early pregnancy [1]. These mice were then used to create mammary epithelial-specific Cyp24a1 knockout in the PyMT mouse breast cancer model (KO) to examine its role in tumour progression. Specific CYP24A1 ablation was confirmed by histology and qPCR analysis. Breast tumour initiation was significantly delayed with palpable tumours occurring at 10 weeks in KO animals (mean=0.140 cm3, SD=0.061 N=17) compared to 7 weeks (mean= 0.385 cm3, SD=0014, N=17) in control animals (CYP24A1 flox/flox Cre- and CYP24A1wt/wt Cre+). At 13 weeks, tumour volume in KO mice was 75% smaller than in control mice (p <0.0001). Furthermore, tumour size at endpoint in KO (6 cm3) was observed at age 18 weeks (mean= 5.6 cm3 SD=0.42 N=19) compared to 13 weeks in control animals (mean= 6.5 cm3 SD=0.22 N=17). Breast tumour number in KO mice (4.6 ± 0.5 N=20) was significantly decreased at death (17-18 weeks) compared to 13-14 weeks control mice (7.9 ± 0.3 N=20). Finally, CYP24A1 knockout animals showed a dramatic reduction in lung metastasis development compared to control animals (2/10 Vs 9/10, P<0.001 N=10). These data suggest inhibiting CYP24A1 is a potential approach to activating the vitamin D pathway in breast cancer prevention and therapy.