Extracellular low phosphate strongly enhances intestinal calcium absorption independently of active vitamin D [1,25(OH)2D3] signaling, but the underlying mechanisms remain poorly characterized. To elucidate the phosphate-dependent regulation of calcium transport, we investigated part of the enteral environment that is involved in 1,25(OH)2D3-independent calcium absorption, which responds to dietary phosphate levels in mice that lack intestinal vitamin D receptor ( Vdr) activity. Impaired calcium absorption in intestinal Vdr-null mice was improved by dietary phosphate restriction. Accordingly, calcium transport in cultured intestinal epithelial cells was increased when the apical side was exposed to low phosphate levels (0.5 mM) compared with normal or high phosphate levels (1.0 or 5.0 mM, respectively). Mechanistically, low phosphate increased ATP in the apical side medium and allowed calcium entry into epithelial cells via the P2X7 purinoreceptor, which results in increased calcium transport. We found that luminal ATP was regulated by the release and degradation of ATP at the epithelium, and phosphate restriction increased ATP release from epithelial cells via connexin-43 hemichannels. Furthermore, ATP degradation by ectonucleotide pyrophosphatase-1 was reduced, which was caused by the reduction of the MAPK cascade. These findings indicate that luminal ATP metabolism regulates transcellular calcium transport in the intestine by an 1,25(OH)2D3-independent mechanism in response to dietary phosphate levels.-Uekawa, A., Yamanaka, H., Lieben, L., Kimira, Y., Uehara, M., Yamamoto, Y., Kato, S., Ito, K., Carmeliet, G., Masuyama, R. Phosphate-dependent luminal ATP metabolism regulates transcellular calcium transport in intestinal epithelial cells.