The intestinal epithelial brush border Na⁺/H⁺ exchanger NHE3 accounts for a large component of intestinal Na absorption. NHE3 is regulated during digestion by signaling complexes on its C-terminus that include the four multi-PDZ domain containing NHERF family proteins. Because the roles of each NHERF appear to vary based on the cell model or intestinal segment studied and because of our recent finding that a NHERF3-NHERF2 heterodimer appears important for NHE3 regulation in Caco-2 cells, we examined the role of NHERF3 and NHERF2 in homozygous C57BL/6 NHERF2 and NHERF3 KO mouse jejunum. The jejunal apical membrane of NHERF3 null mice appeared similar to wild type (WT) in microvillus number and height, which is similar to results previously reported for NHERF2 null mice. NHE3 basal activity was not different from WT in either NHERF2 or NHERF3 null jejunum, while D-glucose stimulated NHE3 activity was reduced in NHERF2 but similar to WT in NHERF3 KO. LPA stimulation and UTP (elevated Ca²⁺) and cGMP inhibition of NHE3 were markedly reduced in both NHERF2 and NHERF3 null jejunum. Forskolin inhibited NHE3 in NHERF3 null jejunum, but the extent of inhibition was reduced compared to WT. The forskolin inhibition of NHE3 in NHERF2 null mice was inconsistent. These results demonstrate similar requirement for NHERF2 and NHERF3 in mouse jejunal NHE3 regulation by LPA, Ca²⁺, and cGMP. The explanation for the similarity is not known but is consistent with involvement of a brush border NHERF3-NHERF2 heterodimer or sequential NHERF dependent effects in these aspects of NHE3 regulation.