Efficient intestinal absorption of dietary vitamin D is required in most people to ensure an adequate status. Thus, we investigated the involvement of ATP binding cassette subfamily B member 1 (ABCB1) in vitamin D intestinal efflux. Both cholecalciferol (D<sub>3</sub>) and 25-hydroxycholecalciferol [25(OH)D<sub>3</sub>] apical effluxes were decreased by chemical inhibition of ABCB1 in Caco-2 cells and increased by ABCB1 overexpression in Griptites or Madin-Darby canine kidney type II cells. Mice deficient for the 2 murine ABCB1s encoded by Abcb1a and Abcb1b genes ( Abcb1<sup>-/-</sup>) displayed an accumulation of 25(OH)D<sub>3</sub> in plasma, intestine, brain, liver, and kidneys, together with an increased D<sub>3</sub> postprandial response after gavage compared with controls. 25(OH)D<sub>3</sub> efflux through Abcb1<sup>-/-</sup> intestinal explants was markedly decreased compared with controls. This reduction of 25(OH)D<sub>3</sub> transfer from plasma to lumen was further confirmed in vivo in intestine-perfused mice. Docking experiments established that both D<sub>3</sub> and 25(OH)D<sub>3</sub> could bind with high affinity to Caenorhabditis elegans P-glycoprotein, used as an ABCB1 model. Finally, in a group of 39 healthy male adults, a single-nucleotide polymorphism (SNP) in ABCB1 (rs17064
) was significantly associated with the fasting plasma 25(OH)D<sub>3</sub> concentration. Thus, we showed here for the first time that ABCB1 is involved in neo-absorbed vitamin D efflux by the enterocytes and that it also contributes to vitamin D transintestinal excretion and likely impacts vitamin D status.-Margier, M., Collet, X., le May, C., Desmarchelier, C., Andre, F., Lebrun, C., Defoort, C., Bluteau, A., Borel, P., Lespine, A., Reboul, E. ABCB1 (P-glycoprotein) regulates vitamin D absorption and contributes to its transintestinal efflux.