2'-Phosphophloretin (2'-PP), a phosphorylated derivative of the plant chalcone, was synthesized. The effect of 2'-PP, on Na⁺-dependent phosphate uptake into intestinal brush-border membrane vesicles (BBMV) isolated from rabbit and rat duodenum and jejunum was examined. 2'-PP decreased Na⁺-dependent phosphate uptake into rabbit BBMV with an IC₅₀ of 55 nM and into rat BBMV with an IC₅₀ of 58 nM. 2'-PP did not affect Na⁺-dependent glucose, Na⁺-dependent sulfate, or Na⁺-dependent alanine uptake by rabbit intestinal BBMVs. 2'-PP inhibition of rabbit intestinal BBMV 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. Binding of [³H]2'-PP to rabbit intestinal BBMV was examined. Binding of [³H]2'-PP was Na⁺-dependent with a K_0.5 for Na⁺(Na⁺ concentration for 50% 2'-PP binding) of 30 mM. The apparent K_s for Na⁺-dependent [³H]2'-PP binding to rabbit BBMVs was 58 nM in agreement with the IC₅₀ for 2'-PP inhibition of Na⁺-dependent phosphate uptake. These results indicate that 2'-PP bound to rabbit or rat intestinal BBMV Na⁺-phosphate cotransporter and inhibited Na⁺-dependent phosphate uptake. In rats treated with 2'-PP by daily gavage, the effect of 2'-PP on serum phosphate, serum glucose, and serum calcium was examined. In a concentration-dependent manner, 2'-PP reduced serum phosphate by 45% 1 wk after starting treatment. 2'-PP did not alter serum calcium or serum glucose. The apparent IC₅₀ for 2'-PP in vivo was 3 µM.