A phosphorylated derivative of the plant chalcone, phloretin, was synthesized. The effect of 2'-phosphophloretin, 2'-PP, on Na⁺-dependent phosphate uptake into intestinal brush border membrane vesicles isolated from rabbit and rat duodenum and jejunum, was examined. 2'-Phosphophloretin (2'-PP) inhibited Na⁺-dependent phosphate uptake with an IC₅₀ of 55 nM in the rabbit and 58 nM in the rat. 2'-PP did not affect Na⁺-dependent glucose or Na⁺-dependent alanine uptake by intestinal brush border membrane vesicles. 2'-PP inhibition of Na⁺-dependent phosphate uptake was sensitive to external phosphate concentration suggesting that 2'-PP inhibition of Na⁺-dependent phosphate uptake was competitive with respect to phosphate. The binding of [^3H] 2'-PP to intestinal brush border membrane vesicles was examined. Binding of [^3H] 2'-PP was Na⁺-dependent with a K_0.5 for Na⁺ of 30 mM. The apparent K_s for Na⁺-dependent [^3H] 2'-PP binding to rabbit brush border membrane vesicles was 48 nM in agreement with the IC₅₀ for 2'-PP inhibition of Na⁺-dependent phosphate uptake. The results indicate that 2'-PP bound to the intestinal Na⁺-/phosphate cotransporter and to selectively inhibit Na⁺-dependent phosphate uptake. The effect of 2'-PP in vivo was examined in rats. In rats treated with 2'-PP by daily gavage the effect of 2'-PP on serum phosphate, calcium and glucose was examined. In a concentration-dependent manner, 2'-PP reduced plasma phosphate 45% one week after starting treatment. 2'-PP did not alter plasma calcium or glucose. The apparent IC₅₀ for 2'-PP in vivo was 3 µM.