Podocyte is the major target in proteinuric kidney disease such as diabetic nephropathy. The underlying molecular mechanisms by which high glucose (HG) results in podocyte damage remain unclear. This study investigated the regulatory role of Smad3, ezrin, and protein kinase A (PKA) in NADPH oxidase (Nox4) expression, reactive oxidative species (ROS) production, and apoptosis in HG-treated podocytes. Human podocyte cell line was cultured and differentiated, then treated with 30 mM HG. Apoptosis and intracellular ROS level was assessed using TUNEL and DCF assay, respectively. Expressions of Nox4, phospho-Smad3 Ser423/425 , phospho-PKA Thr197 , and phospho-ezrin Thr567 were evaluated using Western blotting. ELISA was used to quantify intracellular cAMP concentration and PKA activity. Knockdown assay was used to inhibit the expressions of Smad3, Nox4, and ezrin by lentiviral shRNA. In HG-treated podocytes, the level of phospho-Smad3 Ser423/425 and phospho-ezrin Thr567 was increased significantly, which was accompanied by the reduction of cAMP and phospho-PKA Thr197 . HG-induced apoptosis was significantly prevented by the Smad3 inhibitor SIS3 or shRNA-Smad3. In podocytes expressing shRNA-ezrin or shRNA-Nox4, apoptosis was remarkably mitigated following HG treatment. HG-induced upregulation of phospho-ezrin Thr567 and downregulation of phospho-PKA Thr197 was significantly prevented by SIS3, shRNA-ezrin or shRNA-Smad3. Forskolin, a PKA activator, significantly inhibited HG-mediated upregulation of Nox4 expression, ROS generation, and apoptosis. Additionally, an increase in the ROS level was prohibited in HG-treated podocytes with the knockdown of Nox4, Smad3, or ezrin. Taken together, our findings provided evidence that Smad3-mediated ezrin activation upregulates Nox4 expression and ROS production, by suppressing PKA activity, which may at least in part contribute to HG-induced podocyte apoptosis.