Ouabain, a cardiotonic steroid and a specific inhibitor of the Na(+)-K(+)-ATPase, has been shown to significantly inhibit transcellular Na(+) transport without altering the intracellular Na(+) concentration ([Na(+)](i)) in the epithelial cells derived from the renal proximal tubules. We therefore studied whether ouabain affects the activity and expression of Na(+)/H(+) exchanger isoform 3 (NHE3) representing the major route of apical Na(+) reabsorption in LLC-PK(1) cells. Chronic basolateral, but not apical, exposure to low-concentration ouabain (50 and 100 nM) did not change [Na(+)](i) but significantly reduced NHE3 activity, NHE3 protein, and mRNA expression. Inhibition of c-Src or phosphoinositide 3-kinase (PI3K) with PP2 or wortmannin, respectively, abolished ouabain-induced downregulation of NHE3 activity and mRNA expression. In caveolin-1 knockdown LLC-PK(1) cells, ouabain failed to downregulate NHE3 mRNA expression and NHE3 promoter activity. Ouabain response elements were mapped to a region between -450 and -1,194 nt, where decreased binding of thyroid hormone receptor (TR) and Sp1 to their cognate cis-elements was documented in vitro and in vivo by protein/DNA array analysis, EMSA, supershift, and chromatin immunoprecipitation. These data suggest that, in LLC-PK(1) cells, ouabain-induced signaling through the Na(+)-K(+)-ATPase-Src pathway results in decreased Sp1 and TR DNA binding activity and consequently in decreased expression and activity of NHE3. These novel findings may represent the underlying mechanism of cardiotonic steroid-mediated renal compensatory response to volume expansion and/or hypertension.