Aldosterone (Aldo) plays a direct profibrotic role in the kidney but the underlying mechanism is unclear. We examined the role of Aldo in epithelial-mesenchymal transition (EMT) both in vitro and in vivo. Exposure of human renal proximal tubular cells (HK-2) to Aldo for 48 hours induced EMT as evidenced by conversion to the spindle-like morphology, loss of E-cadherin, and de novo expression of -SMA. The EMT was completely blocked by the selective mineralocorticoid receptor (MR) antagonist eplerenone. Aldo time-dependently increased intracellular reactive oxygen species (ROS) production that was detectable at 15 min and peaked (2.3-fold) at 60 min, as assessed by 2',7'-dichlorofluorescin diacetate fluorescence. Aldo-induced oxidative stress and EMT were both abolished by the mitochondrial respiratory chain complex I inhibitor rotenone, but not the NADPH oxidase inhibitor apocynin. Aldo induced phosphorylation of ERK1/2 that was completely blocked by rotenone. Male 129-C57/BL6 mice were treated with deoxycorticosterone acetate (DOCA)/salt for 3 weeks and animals were treated with vehicle or rotenone (600 ppm in diet) for the last wk. DOCA-salt induced a 2.5-fold increase in -SMA and a 30% reduction of E-cadherin, as assessed by real time RT-PCR, that were both restricted to renal epithelial cells, as determined by immunohistochemistry. In contrast, DOCA-salt-induced changes in -SMA and E-cadherin were completely blocked by treatment with rotenone. These observations suggest that aldosterone induces EMT via MR-mediated, mitochondrial originated-ROS-dependent ERK1/2 activation in renal tubular epithelial cells.