Podocytes are terminally differentiated, actin-rich epithelial cells present on the glomerular basement membrane and critical for glomerular filtration barrier. Injury to podocyte leads to the onset of chronic renal diseases characterized by proteinuria. Well-known mediators of podocyte damage includes transforming growth factor-β (TGF-β) and saturated free fatty acid (FFA), which are increased in kidney tissue level of diabetic patients. Here, we investigated the pathogenic role of mitochondrial reactive oxygen species (ROS) on TGF-β- or FFA-induced podocyte injury in immortalized mouse podocytes. Exogenous TGF-β1 elevated ROS production and podocyte apoptosis through selective upregulation of Nox4. In mouse podocytes, Nox4 was predominantly localized to mitochondria and Nox4 upregulation by TGF-β1 markedly depolarized the mitochondrial membrane potential (Δψm). TGF-β1-induced ROS production and caspase activation were mitigated by an antioxidant, the Nox inhibitor DPI, or siRNA for Nox4. A TGF-β receptor I blocker, SB431542, and knock-down of either Smad2 or Smad3 prevented increase of Nox4 expression, ROS generation, loss of mitochondrial membrane potential, and caspase-3 activation by TGF-β1. Palmitate, as a major saturated FFA, elicited mitochondrial ROS production, Δψm depolarisation, reduced ATP level, and podocyte apoptosis, while a mono-unsaturated FFA oleate itself or in combination with palmitate did not. According to morpho-dynamic studies, palmitate caused not only mitochondrial fragmentation, but also marked ER lumen dilatation. Consistently, palmitate upregulated ER stress proteins such as GRP78/Bip, spliced xbp1, and CHOP. Palmitate depleted the luminal Ca²⁺ level in ER and abolished the cyclopiazonic acid-induced cytosolic Ca²⁺ increase. Palmitate-induced cytotoxicity and ER Ca²⁺ depletion were blocked by a mitochondrial antioxidant, mitoTEMPO. Taken together, these data suggest that mitochondrial dysfunction and/or ER stress in TGF-β- and FFA-induced podocyte apoptosis are mediated by mitochondrial ROS production, which may at least in part contribute to the development and progression of proteinuric glomerular diseases such as diabetic nephropathy.