The primary determinant of circulating 1α,25-dihydroxyvitamin D (1,25[OH](2)D) levels is the activity of 25-hydroxyvitamin D-1α-hydroxylase (cytochrome P450 27B1 [CYP27B1]) in the kidney. Hyperthyroid patients have been reported to have low levels of plasma 1,25(OH)(2)D. However, the detailed mechanism of thyroid hormone action on vitamin D metabolism is still poorly understood. The present study determined whether renal CYP27B1 gene expression was negatively regulated by thyroid hormones. T(3)-induced hyperthyroid mice showed marked decreases in plasma 1,25(OH)(2)D levels and in renal expression of CYP27B1 mRNA but no changes in plasma concentrations of calcium, PTH, or fibroblast growth factor-23. In addition, we observed that T(3) administration significantly decreased plasma 1,25(OH)(2)D and renal CYP27B1 mRNA levels that were increased by low-calcium or low-phosphorus diets and induced hypocalcemia in mice fed a low-calcium diet. Promoter analysis revealed that T(3) decreases the basal transcriptional activity of the CYP27B1 gene through thyroid hormone receptors (TRα and TRβ1) and the retinoid X receptor α (RXRα) in renal proximal tubular cells. Interestingly, we identified an everted repeat negative thyroid hormone response element (1α-nTRE) overlapping the sterol regulatory element (1α-SRE) and the TATA-box -50 to -20 base pairs from the human CYP27B1 gene transcription start site. Finally, we established that CYP27B1 gene transcription is positively regulated by SRE-binding proteins and that a T(3)-bound TRβ1/RXRα heterodimer inhibits SRE-binding protein-1c-induced transcriptional activity through the 1α-nTRE. These results suggest that transcriptional repression of the CYP27B1 gene by T(3)-bound TRs/RXRα, acting through the 1α-nTRE, results in decreased renal CYP27B1 expression and plasma 1,25(OH)(2)D levels.