To study the role of the epithelial calcium channel TRPV6 and the calcium binding protein calbindin-D_9k in intestinal calcium absorption, TRPV6 knock out (KO), calbindin-D_9k KO, and TRPV6/calbindin-D_9k double knock out (DKO) mice were generated. TRPV6 KO, calbindin-D_9k KO, and TRPV6/calbindin-D_9k DKO mice have serum calcium levels similar to those of wild type (WT) mice (10 mg Ca⁺⁺/dl). In the TRPV6 KO and the DKO mice, however, there is a 1.8 fold increase in serum PTH levels (p<0.05 compared to WT). Active intestinal calcium transport was measured using the everted gut sac method. Under low dietary calcium conditions there was a 4.1, 2.9, and 3.9 fold increase in calcium transport in the duodenum of WT, TRPV6 KO, and calbindin-D_9k KO mice, respectively (n=8–22/group; p>0.1 WT vs. calbindin-D_9k KO and p<0.05 WT vs. TRPV6 KO on the low calcium diet). Duodenal calcium transport was increased 2.1 fold in the TRPV6/calbindin-D_9k DKO mice fed the low calcium diet (p<0.05 WT vs. DKO). Active calcium transport was not stimulated by low dietary calcium in the ileum of the WT or KO mice. 1,25(OH)₂D₃ administration to vitamin D deficient nullmutant and WT mice also resulted in a significant increase in duodenal calcium transport (1.4 – 2.0 fold, p<0.05 compared to vitamin D deficient mice). This study provides evidence for the first time using nullmutant mice that significant active intestinal calcium transport occurs in the absence of TRPV6 and calbindin-D_9k, thus challenging the dogma that TRPV6 and calbindin-D_9k are required for vitamin D induced active intestinal calcium transport.