The expression of CYP27B1 or vitamin D 1α-hydroxylase (1α-OHase) and CYP24A1 in specific tissues may act as the central part between 25-hydroxyvitamin D [25(OH)D] serum levels and the anticancer effects of1α,25-dihydroxyvitamin D [1α,25(OH)2D3],alternative splicing of these enzymes may affect their biological functions. Here, we describe the expression of CYP24A1 and its splicing variants detected in breast cells and tissues. Manifestation of CYP24A1 mRNA was measured by RT-PCR followed by western blot analysis for protein expression. In MCF-7 cells, the expression of CYP24A1 protein was reduced by about 57% compared to MCF-10F cells. Western blot analysis revealed a signal band at 56 kDa, with additional bands detected at 42 and 44 kDa. The expression of CYP24A1 mRNA was reduced by about 58% in breast cancer tissues. We found only one signal in the benign tissues at 56 kDa in western blot, whereas in malignant tissue, an additional band was detected at 40kDa. Our results suggest that alternative splicing of CYP24A1 may lead to a catalytically dysfunctional enzyme.