The long-term effects of ouabain on transepithelial Na⁺ transport involve transcriptional down-regulation of apical NHE3 (Sodium/hydrogen exchanger, isoform 3). The aim of this study is to determine whether ouabain could acutely regulate NHE3 via a post-transcriptional mechanism in LLC-PK1 cells. We observed that basolateral, but not apical, application of ouabain for 1h significantly reduced transepithelial Na⁺ transport. This effect was not due to changes in the integrity of tight junctions or increases in intracellular Na⁺ concentration. Ouabain regulated the trafficking of NHE3 and subsequently inhibited its activity, a process independent of intracellular Na⁺ concentration. Ouabain-induced NHE3 trafficking was abolished by either cholesterol depletion or Src inhibition. Moreover, ouabain increased the intracellular Ca²⁺ concentration. Pre-treatment of cells with the intracellular Ca²⁺ chelator BAPTA-AM blocked ouabain-induced trafficking of NHE3. Also, blocking Na/K-ATPase endocytosis by a PI3K inhibitor was equally effective in attenuating ouabain-induced NHE3 trafficking. These data indicate that ouabain acutely stimulates NHE3 trafficking by activating the basolateral Na/K-ATPase signaling complex. Taken together with our previous observations, we propose that ouabain can simultaneously regulate basolateral Na/K-ATPase and apical NHE3, leading to inhibition of transepithelial Na⁺ transport. This mechanism may be relevant to proximal tubular sodium handling during conditions associated with increases in circulating endogenous cardiotonic steroids.