Genetic deletion or inflammation-associated downregulation of the PDZ-adaptor PDZK1/NHERF3 results in a decreased transport activity of the Na+/H+ exchanger NHE3 in the murine intestine, accompanied by increased enterocyte NHE3 mRNA expression levels, but a borderline reduced NHE3 protein expression in or near the apical plasmamembrane (Hillesheim et al. Pflüger’s Arch 2007, Cinar et al. J. Physiol. 2007, Lenzen et al. Plos One 2012). This suggested that PDZK1 may play a role in targeting/stabilizing NHE3 in the enterocyte brush border membrane. To answer this question, we searched for an intestinal cell line that endogenously expresses PDZK1 and found the spontaneously differentiating cell line Caco-2BBe to have robust endogenous PDZK1 expression. VSVG-tagged NHE3 was stably expressed in Caco-2BBe cells (Caco-2BBe/NHE3-VSVG), followed by stable knock donw of PDZK1 by using recombinant lentiviral sh-RNA transfer. We then studied the effect of PDZK1 knockdown on membrane NHE3 half life, brush border membrane expression, and transport function of NHE3. Biotinylation of membrane proteins, followed by Western analysis and immunocytochemistry revealed a significantly decreased amount of apical membrane NHE3 in PDZK1 knock down (KD) cells, with more cytoplasmic/endosomal and basolaterally localized NHE3, compared to control cells. This correlated with a significantly decreased acid-activated NHE3 transport activity in the PDZK1 KD compared to control cells. Membrane half life studies for NHE3 were performed by following the degradation of biotinylated NHE3 over time. Our study revealed that that control cells have a biphasic time course for NHE3 membrane loss, with an early rapid phase followed by a slower phase with a linear loss of the ensuing 48 hours. In PDZK1 KD cells, the initial percentage of NHE3 that was expressed in the membrane was significantly lower than in control cells, and the early phase of rapid loss of membrane NHE3 was completely ablated while the second, slower phase was identical to control cells. Conclusions: The PDZ-adaptor protein PDZK1 is important for the targeting and/or retention of the Na+/H+ exchanger isoform 3 to the brush border membrane of intestinal epithelial cells. The data further suggest that human NHE3 is retrieved from the membrane and degraded by different pathways with different time constants and differentially requiring PDZ-adaptor proteins. Further work is necessary to characterize the protein complex formation that decides the pathway of NHE3 into recycling endosomes versus lysosomal/proteasomal degradation.