Estrogen treatment causes renal phosphate (Pi) wasting and hypophosphatemia in rats and humans; however the signaling mechanisms mediating this effect are still poorly understood. To determine the specific roles of estrogen receptor isoforms (ERα and ERβ) and Klotho pathway in mediating these effects, we studied the effects of estrogen on renal Pi handling in female mice with null mutations of ERα or ERβ or Klotho and their wild-type (WT) using balance studies in metabolic cages. Estrogen treatment of WT and ERβ KO mice caused a significant reduction in food intake along with increased renal phosphate wasting. The later resulted from a significant downregulation of NaPi-IIa and NaPi-IIc protein abundance. The mRNA expression levels of both transporters were unchanged in estrogen-treated mice. These effects on both food intake and renal Pi handling were abolished in ER KO mice. Estrogen treatment of Klotho KO mice or PTH-depleted thyro-parathyroidectomized mice exhibited a significant downregulation of NaPi-IIa with no change in the abundance of NaPi-IIc. Estrogen treatment of a cell line (U20S) stably co-expressing both ERα and ERβ caused a significant downregulation of NaPi-IIa protein, when transiently transfected with a plasmid containing full-length or ORF-3'UTR but not 5'UTR-ORF of mouse NaPi-IIa transcript. Conclusions: estrogen causes phosphaturia and hypophosphatemia in mice. These effects result from downregulation of NaPi-IIa and NaPi-IIc proteins in the proximal tubule through the activation of ERα. The downregulation of NaPi-IIa by estrogen involves 3'UTR of its mRNA and is independent of Klotho/fgf23 and PTH signaling pathways.