As glucose-stimulated insulin secretion of pancreatic β cell is triggered and promoted by the metabolic messengers derived from mitochondria, mitochondria take a central stage in the normal function of β cells. β cells in diabetics were chronically exposed to hyperglycemia stimulation, which have been reported to exert deleterious effects on β-cell mitochondria. However, the mechanism of the toxic effects of hyperglycemia on β-cell mitochondria was not clear. In this study, we characterized the biological functional changes of rat INS-1β cells and their mitochondria with chronic exposure to hyperglycemia and created a research model of chronic hyperglycemia-induced dysfunctional β cells with damaged mitochondria. Then, SILAC-based quantitative proteomic approach was used to compare the mitochondrial protein expression from high glucose treated INS-1β cells and control cells. The expression of some mitochondrial proteins was found with significant changes. Functional classification revealed most of these proteins were related with oxidative phosphorylation, mitochondrial protein biosynthesis, substances metabolism, transport, and cell death. These results presented some useful information about the effect of glucotoxicity on the β-cell mitochondria. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.