The sodium-dependent renal phosphate transporter (Npt2, Na-Pi IIa) is the major regulated phosphate transporter in the renal proximal convoluted tubule. Npt2 associates with a number of PDZ-containing proteins including Na⁺H⁺ exchanger regulatory factor-1 (NHERF-1). To determine whether NHERF-1 is involved in the acute regulation of phosphate transport, wild-type and NHERF-1 (–/–) mice were stabilized on a high-phosphate diet and then acutely changed to a low-phosphate diet. At 24 h after the change to a low-phosphate diet, there was a significant decrease in the urinary excretion of phosphate in both groups but the urinary excretion of phosphate in NHERF-1 (–/–) mice was significantly higher than in wild-type animals (1,097 ± 356 vs. 255 ± 54 ng/min, P < 0.05). Renal mRNA levels and total cellular Npt2 protein did not differ between the animal groups or in response to the changes in diet. Renal brush-border membrane (BBM) expression of Npt2 protein, however, was lower in NHERF-1 (–/–) mice compared with wild-type. In addition, with both the high- and low-phosphate diets, there was increased detection of Npt2 in submicrovillar domains that were particularly prominent in NHERF-1 (–/–) mice compared with wild-type animals. On the other hand, a change from a low-phosphate diet to a high-phosphate diet was associated with a similar increase in the urinary excretion of phosphate in wild-type and NHERF-1 (–/–) animals. These experiments demonstrate that full renal adaptation to a low-phosphate diet requires NHERF-1, which serves to increase BBM expression of Npt2.