Proteins expressing postsynaptic density (PSD)-95/ Drosophila disk large (Dlg)/zonula occludens-1 (ZO-1) (PDZ) domains are commonly involved in moderating receptor, channel, and transporter activities at the plasma membrane in a variety of cell types. At the apical membrane of renal proximal tubules (PT), the type IIa NaPi cotransporter (NaPi-IIa) binds specific PDZ domain proteins. Shank2E is a spliceoform of a family of PDZ proteins that is concentrated at the apical domain of liver and pancreatic epithelial cell types and is expressed in kidney. In the present study, immunoblotting of enriched plasma membrane fractions and immunohistology found Shank2E concentrated at the brush border membrane of rat PT cells. Confocal localization of Flag-Shank2E and enhanced green fluorescent protein-NaPi-IIa in cotransfected OK cells showed these proteins colocalized in the apical microvilli of this PT cell model. Shank2E coimmunoprecipitated with NaPi-IIa from rat renal cortex tissue and HA-NaPi-IIa coprecipitated with Flag-Shank2E in cotransfected human embryonic kidney HEK cells. Domain analysis showed that the PDZ domain of Shank2E specifically bound NaPi-IIa and truncation of the COOH-terminal TRL motif from NaPi-IIa abolished this binding, and Far Western blotting showed that the Shank2E- NaPi-IIa interaction occurred directly between the two proteins. NaPi-IIa activity is regulated by moderating its abundance in the apical membrane. High-Pi conditions induce NaPi-IIa internalization and degradation. In both rat kidney PT cells and OK cells, shifting to high-Pi conditions induced an acute internal redistribution of Shank2E and, in OK cells, a significant degree of degradation. In sum, Shank2E is concentrated in the apical domain of renal PT cells, specifically binds NaPi-IIa via PDZ interactions, and undergoes Pi-induced internalization.