Background: Peroxidation of renal cells is a critical event in the nucleation and formation of calcium oxalate crystals under hyperoxaluric conditions. We previously demonstrated that oxalate-induced peroxidative injury is one of the major mechanisms in promoting crystal attachment to renal epithelial cells. Methods: In this study we have demonstrated that the mechanism of oxalate-induced peroxidative injury is through the induction of TGF-β<sub>1</sub> and glutathione (GSH) redox imbalance in LLC-PK<sub>1</sub> cells. Results: LLC-PK<sub>1</sub>, renal epithelial cells exposed to oxalate had significantly higher reactive oxygen species (ROS) production; higher TGF-β<sub>1</sub> levels, as measured by ELISA (1.89 ± 0.035 fold increase) or Western blot (1.65 ± 0.01 fold increase); increased malondialdehyde formation; increased LDH release, and loss of cell viability. In addition, oxalate exposure significantly decreased GSH content, glutathione reductase, glucose-6-phosphate dehydrogenase activities, and increased oxidized GSH content. Treatment with vitamin E, neutralizing anti-TGF-β antibody, or diphenylene iodium, an inhibitor of NAD(P)H oxidase, significantly inhibited oxalate-induced ROS production and prevented peroxidative injury and cytolysis. Vitamin E, catalase, or desferoxamine treatment also significantly restored the oxalate-induced cellular GSH redox status toward the control level, and vitamin E treatment significantly attenuated the oxalate-mediated increase in TGF-β<sub>1</sub> protein in cultured LLC-PK<sub>1</sub> cells. Conclusions: This is the first study to demonstrate that the mechanism of oxalate-induced free radical production in renal tubular epithelial cells is through the activation of NAD(P)H oxidase via cytokine TGF-β<sub>1</sub> induction. These results also provide direct evidence that antioxidant therapy might prevent calcium oxalate nucleation and kidney stone formation by preventing oxalate-mediated peroxidative injury and GSH redox imbalance.