Vitamin D plays a critical role in tissue homeostasis by regulating the expression of genes affecting cell proliferation, differentiation, and apoptosis. The vitamin D 24-hydroxylase CYP24A1 functions in vitamin D target tissues to degrade the hormonal form of vitamin D. Existing knowledge regarding dysregulated CYP24A1 expression supports its candidacy as a putative oncogene. Here, we found that the suppression of constitutive CYP24A1 expression conferred target cells with increased susceptibility to apoptosis and consequently inhibited anchorage-independent growth in breast carcinoma cells. In addition, suppression of vitamin D metabolism following knockdown of CYP24A1 significantly reduced tumor growth in vivo. These data provide substantial evidence for a pro-survival and stimulatory oncogenic effect of CYP24A1 in breast carcinoma cells.