The epithelial Ca²⁺ channels TRPV5 and TRPV6 are localized to the brush border membrane of intestinal cells and constitute the postulated rate-limiting entry step of active Ca²⁺ absorption. The aim of the present study was to investigate the hormonal regulation of these channels. To this end, the effect of 17β-estradiol (17β-E₂), 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], and dietary Ca²⁺ on the expression of the duodenal Ca²⁺ transport proteins was investigated in vivo and analyzed using realtime quantitative PCR. Supplementation with 17β-E₂ increased duodenal gene expression of TRPV5 and TRPV6 but also calbindin-D_9K and plasma membrane Ca²⁺-ATPase (PMCA1b) in ovariectomized rats. 25-Hydroxyvitamin D₃-1α-hydroxylase (1α-OHase) knockout mice are characterized by hyperparathyroidism, rickets, hypocalcemia, and undetectable levels of 1,25(OH)₂D₃ and were used to study the 1,25(OH)₂D₃-dependency of the stimulatory effects of 17β-E₂. Treatment with 17β-E₂ upregulated mRNA levels of duodenal TRPV6 in these 1α-OHase knockout mice, which was accompanied by increased serum Ca²⁺ concentrations from 1.69 ± 0.10 to 2.03 ± 0.12 mM (P < 0.05). In addition, high dietary Ca²⁺ intake normalized serum Ca²⁺ in these mice and upregulated expression of genes encoding the duodenal Ca²⁺ transport proteins except for PMCA1b. Supplementation with 1,25(OH)₂D₃ resulted in increased expression of TRPV6, calbindin-D_9K, and PMCA1b and normalization of serum Ca²⁺. Expression levels of duodenal TRPV5 mRNA are below detection limits in these 1α-OHase knockout mice, but supplementation with 1,25(OH)₂D₃ upregulated the expression to significant levels. In conclusion, TRPV5 and TRPV6 are regulated by 17β-E₂ and 1,25(OH)₂D₃, whereas dietary Ca²⁺ is positively involved in the regulation of TRPV6 only.