Ezrin is an actin-binding protein that crosslinks between the plasma membrane proteins (adhesion proteins, transporters, channels) and actin cytoskeleton. It is involved in microvilli formation, membrane fusion and protein trafficking. Ezrin is highly expressed in stomachs, small intestines, and kidney. In the kidney, it is mainly localized at brush border membrane (BBM) of proximal tubules, where phosphate (Pi) is reabsorbed by Na+/Pi cotransporters. There, ezrin is reported to be associated with a major Na+/Pi cotransporter, Npt2a, through a scaffolding protein, Na+/H+ exchanger regulatory factor (NHERF) 1. In the present study, we focused on the physiological role of ezrin in the renal Pi reabsorption and duodenal Ca2+ absorption by using ezrin knockdown (Vil2kd/kd) mice in which expression of ezrin was down-regulated as low as 7% compared with wild-type (WT) mice (Tamura et al. 2005). We found that Vil2kd/kd mice exhibited hypophosphatemia and hypocalcemia. The reduced plasma concentration of Pi was attributed to the defective urinary Pi reabsorption. Immunofluorescence staining revealed that cell surface expression of Npt2a and NHERF1 was impaired in the kidney of Vil2kd/kd mice. Immunoblotting also revealed that the Npt2a and NHERF1 expressions at the BBM fractions of proximal tubules were markedly reduced in the kidney of Vil2kd/kd mice. On the other hand, urinary loss of Ca2+ was not observed in Vil2kd/kd mice. Plasma concentration of 1,25-dihydroxyvitamin D was elevated following reduced plasma Pi level, and mRNA level of vitamin D-dependent TRPV6 Ca2+ channel was increased in the duodenum of Vil2kd/kd mice. However, the expression of TRPV6 at apical membrane was disturbed in Vil2kd/kd mice due to ezrin knockdown. Vil2kd/kd mice also exhibited osteomalacia. Bone mineral density in tibiae was significantly lowered in the adult and young Vil2kd/kd mice compared with WT mice. These results strongly suggest that ezrin is required for the regulation of systemic Pi and Ca2+ homeostasis in vivo.