Key points: Intestinal absorption of phosphate proceeds via an active/transcellular route mostly mediated by NaPi-IIb/Slc34a2 and a poorly characterized passive/paracellular pathway. Intestinal phosphate absorption and expression of NaPi-IIb are stimulated by 1,25(OH)2 vitamin D3 but whether NaPi-IIb is the only target under hormonal control remains unknown. We report that administration of 1,25(OH)2 vitamin D3 to wild-type mice resulted in the expected increase in active transport of phosphate in jejunum, without changing paracellular fluxes. Instead, the same treatment failed to alter phosphate transport in intestinal-depleted Slc34a2-deficient mice. In both genotypes, 1,25(OH)2 vitamin D3 induced similar hyperphosphaturic responses and changes in the plasma levels of FGF23 and PTH. While urinary phosphate loss induced by administration of 1,25(OH)2 vitamin D3 did not alter plasma phosphate, further studies should investigate whether chronic administration would lead to phosphate imbalance in mice with reduced active intestinal absorption. Intestinal absorption of phosphate is stimulated by 1,25(OH)2 vitamin D3. At least two distinct mechanisms underlie phosphate absorption in the gut, an active transcellular transport requiring the Na+ /phosphate cotransporter NaPi-IIb/Slc34a2, and a poorly characterized paracellular passive pathway. 1,25(OH)2 vitamin D3 stimulates NaPi-IIb expression and function, and loss of NaPi-IIb reduces intestinal phosphate absorption. However, it is remains unknown whether NaPi-IIb is the only target for hormonal regulation by 1,25(OH)2 vitamin D3 . Here we compared the effects of intraperitoneal administration of 1,25(OH)2 vitamin D3 (2 days, once per day) in wild-type and intestinal-specific Slc34a2-deficient mice, and analysed trans- vs. paracellular routes of phosphate absorption. We found that treatment stimulated active transport of phosphate only in jejunum of wild-type mice, though NaPi-IIb protein expression was upregulated in jejunum and ileum. In contrast, 1,25(OH)2 vitamin D3 administration had no effect in Slc34a2-deficient mice, suggesting that the hormone specifically regulates NaPi-IIb expression. In both groups, 1,25(OH)2 vitamin D3 elicited the expected increase of plasma fibroblast growth factor 23 (FGF23) and reduction of parathyroid hormone (PTH). Treatment resulted in hyperphosphaturia (and hypercalciuria) in both genotypes, though mice remained normophosphataemic. While increased intestinal absorption and higher FGF23 can trigger the hyperphosphaturic response in wild types, only higher FGF23 can explain the renal response in Slc34a2-deficient mice. Thus, 1,25(OH)2 vitamin D3 stimulates intestinal phosphate absorption by acting on the active transcellular pathway mostly mediated by NaPi-IIb while the paracellular pathway appears not to be affected.