Podocytes are terminally differentiated, actin-rich epithelial cells present on the glomerular basement membrane and critical for glomerular filtration process. Podocyte injury leads to onset of chronic renal diseases characterized by proteinuria. Elevated level of TGFβ in pathogenic condition is associated with podocyte damage ultimately resulting in apoptosis and detachment. The underlying mechanism of apoptosis, however, remains not clearly understood in these cells. We investigated pathogenic mediators for proapoptotic effects of TGFβ in cultured immortalized mouse podocytes. TGFβ induced podocyte apoptosis was mediated by elevated level of reactive oxygen species (ROS) which was blocked by N-acetyl-cystein, an antioxidant. Exogenous TGFβ1 selectively increased the expression of only Nox4, excluding other NADPH oxidase (NOX) enzymes. Consistent with Nox4 upregulation, the total NOX activity was higher in TGFβ1-treated podocytes. TGFβ1 enhanced total ROS and elicited apoptosis indicated by the increased number of apoptotic nuclei and elevated level of cleaved caspase-3. These changes by TGFβ1 were blocked by SB431542 and DPI, a potent inhibitor of Type I TGFβ receptor and a selective inhibitor of NADPH oxidases, respectively. Knock-down of Nox4 by siRNA (siNOX4) treatment blocked TGFβ1-induced ROS production and caspase activation. In podocyte, Nox4 was localized to mitochondria as it co-localized with mitochondrial protein Cox1. TGFβ1 elicited mitochondrial membrane depolarization which was recovered by siNOX4 or DPI treatment. In addition, siRNA for smad2 or smad3 reduced Nox4 in transcript and protein level, total ROS generation, and cleaved caspase-3 activation. These results suggest that TGFβ-induced activation of Smad2/3 is responsible for Nox4 up-regulation, and ROS production by NOX4 closely participates in TGFβ-induced apoptosis via mitochondrial dysfunction which might be a crucial mechanism for the development and progression of proteinuric glomerular diseases including diabetic nephropathy.