Our previous studies showed that renal proximal tubular cells (RPTC) express Ca(2+)-independent phospholipase A(2)gamma (iPLA(2)gamma) in endoplasmic reticulum (ER) and mitochondria and that iPLA(2)gamma prevents and/or repairs lipid peroxidation induced by oxidative stress. Our present studies determined the importance of iPLA(2)gamma in mitochondrial and cell function using an iPLA(2)gamma-specific small hairpin ribonucleic acid (shRNA) adenovirus. iPLA(2)gamma expression and activity were decreased in the ER by 24 h and in the mitochondria by 48 h compared with scrambled shRNA adenovirus-treated cells. Lipid peroxidation was elevated by 2-fold at 24 h and remained elevated through 72 h in cells with decreased iPLA(2)gamma. Using electrospray ionization-mass spectrometry, primarily phosphatidylcholines and phosphatidylethanolamines were increased in iPLA(2)gamma-shRNA-treated cells. At 48 h after exposure to the iPLA(2)gamma shRNA, uncoupled oxygen consumption was inhibited by 25% and apoptosis was observed at 72 and 96 h. RPTC with decreased iPLA(2)gamma expression underwent apoptosis when exposed to a nonlethal concentration of the oxidant tert-butyl hydroperoxide (TBHP). Exposure of control cells to a nonlethal concentration of TBHP induced iPLA(2)gamma expression in RPTC. These results suggest that iPLA(2)gamma is required for the prevention and repair of basal lipid peroxidation and the maintenance of mitochondrial function and viability, providing further evidence for a cytoprotective role for iPLA(2)gamma from oxidative stress.