Initiation of Na(+)-glucose cotransport in intestinal epithelial cells leads to activation of the apical Na(+)-H(+) exchanger NHE3 and subsequent increases in cytoplasmic pH (pH(i)). This process requires activation of p38 mitogen-activated protein (MAP) kinase, but additional signaling intermediates have not been identified. One candidate is the cytoskeletal linker protein ezrin, which interacts with NHE3 via specific regulatory proteins. The data show that initiation of Na(+)-glucose cotransport resulted in rapid increases in both apical membrane-associated NHE3 and cytoskeletal-associated ezrin and occurred in parallel with ezrin phosphorylation at threonine 567. Phosphorylation at this site is known to activate ezrin and increase its association with actin. Consistent with a central role for ezrin activation in this NHE3 regulation, an N-terminal dominant negative ezrin construct inhibited both NHE3 recruitment and pH(i) increases after Na(+)-glucose cotransport. Ezrin phosphorylation occurred in parallel with p38 MAP kinase activation, and the latter proceeded normally in cells expressing dominant negative ezrin. In contrast, inhibition of p38 MAP kinase prevented increases in ezrin phosphorylation after initiation of Na(+)-glucose cotransport. Thus, ezrin phosphorylation after Na(+)-glucose cotransport requires p38 MAP kinase activity, but p38 MAP kinase activation does not require ezrin function. These data describe a specific role for ezrin in the coordinate regulation of Na(+)-glucose cotransport and Na(+)-H(+) exchange. Intact ezrin function is necessary for NHE3 recruitment to the apical membrane and NHE3-dependent pH(i) increases triggered by Na(+)-glucose cotransport. The data also define a pathway of p38 MAP kinase-dependent ezrin activation.