Transient receptor potential vanilloid 6 (TRPV6) channels play an important role in intestinal Ca²⁺ transport. These channels undergo Ca²⁺-induced inactivation. Here we show that Ca²⁺ flowing through these channels activates phospholipase C (PLC) leading to the depletion of phosphatidylinositol 4,5-bisphosphate (PIP₂) and formation of inositol 1,4,5-trisphosphate in TRPV6-expressing cells. PIP₂ depletion was inhibited by the two structurally different PLC inhibitors 1-[6-[[17β-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) and edelfosine. Ca²⁺-induced inactivation of TRPV6 was also prevented by the PLC inhibitors in whole-cell patch-clamp experiments. Ca²⁺ signals in TRPV6-expressing cells were transient upon restoration of extracellular Ca²⁺ but were rendered more sustained by the PLC inhibitors. Finally, intestinal Ca²⁺ transport in the everted duodenal sac assay was enhanced by edelfosine. These observations suggest that Ca²⁺-induced inactivation of TRPV6 limits intestinal Ca²⁺ absorption and raise the possibility that Ca²⁺ absorption can be enhanced pharmacologically by interfering with PLC activation.