Abstract Epithelial-to-mesenchymal transition (EMT) is an important developmental process, participates in tissue repair, and occurs during pathologic processes of tumor invasiveness, metastasis, and tissue fibrosis. The molecular mechanisms leading to EMT are poorly understood. Although it is well documented that transforming growth factor (TGF)-β plays a central role in the induction of EMT, the targets of TGF-β signaling are poorly defined. We have shown earlier that Na,K-ATPase β1-subunit levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. In this study, we provide evidence that Na,K-ATPase is a new target of TGF-β1–mediated EMT in renal epithelial cells, a model system used in studies of both cancer progression and fibrosis. We show that following treatment with TGF-β1, the surface expression of the β1-subunit of Na,K-ATPase is reduced, before well-characterized EMT markers, and is associated with the acquisition of a mesenchymal phenotype. RNAi-mediated knockdown confirmed the specific involvement of the Na,K-ATPase β1-subunit in the loss of the epithelial phenotype and exogenous overexpression of the Na,K-ATPase β1-subunit attenuated TGF-β1–mediated EMT. We further show that both Na,K-ATPase α- and β-subunit levels are highly reduced in renal fibrotic tissues. These findings reveal for the first time that Na,K-ATPase is a target of TGF-β1–mediated EMT and is associated with the progression of EMT in cancer and fibrosis. Mol Cancer Ther; 9(6); 1515–24. ©2010 AACR.