Chronic hyperglycemia leads to deterioration of insulin release from pancreatic β-cells as well as insulin action on peripheral tissues. However, the mechanism underlying β-cell dysfunction resulting from glucose toxicity has not been fully elucidated. The aim of the present study was to define a set of alterations in mitochondrial protein profiles of pancreatic β-cell line in response to glucotoxic condition using 2-DE and tandem mass spectrometry. INS1E cells were incubated in the presence of 5.5 and 20 mM glucose for 72 hrs and mitochondria were isolated. Approximately 75 protein spots displayed significant changes (p < 0.05) in relative abundance in the presence of 20 mM glucose compared to controls. Mitochondrial proteins down regulated under glucotoxic conditions includes ATP synthase α chain and δ chain, malate dehydrogenase, aconitase, trifunctional enzyme β subunit, NADH cytochrome b5 reductase and voltage-dependent anion-selective channel protein (VDAC) 2. VDAC1, 75 kDa glucose-regulated protein, heat shock protein (HSP) 60 and HSP10 were found to be upregulated. The orchestrated changes in expression of VDACs and multiple other proteins involved in nutrient metabolism, ATP synthesis, cellular defense, glycoprotein folding and mitochondrial DNA stability may explain cellular dysfunction in glucotoxicity resulting in altered insulin secretion.