The cytochrome P450 25-hydroxyvitamin D3-1alpha-hydroxylase (CYP27b1) plays a pivotal role in vitamin D physiology by catalyzing synthesis of active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In common with other P450s, CYP27b1 is known to exhibit alternative splicing. Here we have cloned and sequenced several novel intron 2-containing, noncoding splice variant mRNAs for CYP27b1 in 1,25(OH)2D3-producing HKC-8 human proximal tubule and THP-1 monocytic cells. Regulation of 1,25(OH)2D3 synthesis in these cell lines by calciotropic and noncalciotropic factors was associated with altered expression of the CYP27b1 splice variants. To assess the functional significance of this, HKC-8 cells were transfected with short hairpin RNA (shRNA) to inhibit mRNAs containing sequences from intron 2. This resulted in a significant increase in the expression of CYP27b1 protein and synthesis of 1,25(OH)2D3 by HKC-8 cells compared with control cells for two different intron 2-containing shRNAs (both P<0.001). shRNA to intron 2 had no significant effect on the levels of wild-type CYP27b1 mRNA, suggesting a posttranscriptional mechanism of action. By contrast, shRNA to wild-type CYP27b1 suppressed transcription and activity of the enzyme by 70 and 31%, respectively (both P<0.01). These data indicate that noncoding splice variants of CYP27b1 are functionally active and may play a significant role in the regulation of 1,25(OH)2D3 synthesis during normal physiology.