unassigned: C-peptide, produced by pancreatic β cells and co-secreted in the bloodstream with insulin, has antioxidant properties in glucose- and hydrogen peroxide (HO)-exposed INS1 β cells. Palmitic acid, the most physiologically abundant long-chain free fatty acid in humans, is metabolized in peroxisomes of β cells accumulating HO that can lead to oxidative stress. Here, we tested the hypothesis that C-peptide protects β cells from palmitic acid-induced stress by lowering peroxisomal HO. unassigned: We exposed INS1 β cells to palmitic acid and C-peptide in the setting of increasing glucose concentration and tested for changes in parameters of stress and death. To study the ability of C-peptide to lower peroxisomal HO, we engineered an INS1 β cell line stably expressing the peroxisomal-targeted HO sensor HyPer, whose fluorescence increases with cellular HO. An INS1 β cell line stably expressing a live-cell fluorescent catalase reporter was used to detect changes in catalase gene expression. unassigned: C-peptide protects INS1 β cells from the combined effect of palmitic acid and glucose by reducing peroxisomal HO to baseline levels and increasing expression of catalase. unassigned: In conditions of glucolipotoxicity, C-peptide increases catalase expression and reduces peroxisomal oxidative stress and death of INS1 β cells. Maintenance of C-peptide secretion is a pro-survival requisite for β cells in adverse conditions. Loss of C-peptide secretion would render β cells more vulnerable to stress and death leading to secretory dysfunction and diabetes.