The PDZ‐binding protein PDZK1 (NHERF3/CAP70/PDZ‐dc‐1) in vitro binds to NHE3, but its role in the regulation of NHE3 activity in native enterocytes is unknown. This study was undertaken to understand the physiological role of PDZK1 in regulating NHE3 activity in native murine colonic enterocytes. NHE3 transport rates were assessed fluorometrically in BCECF‐loaded colonic crypts in the NHE3‐expressing cryptal openings by measuring acid‐activated, Na⁺‐dependent, Hoe 642‐insensitive proton efflux rates. NHE3 mRNA expression levels and NHE3 total enterocyte and brush border membrane (BBM) protein abundance were determined by quantitative PCR and Western analysis and immunohistochemistry. In pdzk1−/− colonic surface cells, acid‐activated NHE3 transport rates were strongly reduced, and the inhibitory effect of forskolin and ionomcyin was virtually abolished. Hyperosmolarity, on the other hand, still had an inhibitory effect. In addition, the NHE3‐selective inhibitor S1611 inhibited acid‐activated NHE3 activity in pdzk1−/− and +/+ mice, suggesting that functional NHE3 is present in pdzk1‐deficient colonocytes. NHE1 and NHE2 activity was not altered in pdzk1−/− colonic crypts. Immunohistochemistry revealed apical NHE3 staining in pdzk1−/− and +/+ proximal colon, and Western blot analysis revealed no difference in NHE3 abundance in colonic enterocyte homogenate as well as brush border membrane. Lack of the PDZ‐adaptor protein PDZK1 in murine proximal colonic enterocytes does not influence NHE3 abundance or targeting to the apical membrane, but abolishes NHE3 regulation by cAMPergic and Ca²⁺ ‐dependent pathways. It leaves NHE3 inhibition by hyperosmolarity intact, suggesting an important and selective role for PDZK1 in the agonist‐mediated regulation of intestinal NHE3 activity.