The cytochrome P450 25-hydroxyvitamin D₃-1-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, non-coding 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 non-calciotropic factors was associated with altered expression of the CYP27b1 splice variants. To assess the functional significance of this, HKC-8 cells were transfected with 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 to 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 non-coding 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.