It has been proposed that mitochondrial dysfunction is involved in the pathogenesis of type 2 diabetes (T2D). To dissect the underlying mechanisms, we performed a multiplexed proteomics study on liver mitochondria isolated from a spontaneous diabetic rat model before/after they are rendered diabetic. All together we identified 1091 mitochondrial proteins, 228 phosphoproteins and 355 hydroxyproteins. Mitochondrial proteins were found to undergo expression changes in a highly correlated fashion during T2D development. For example, proteins involved in ß-oxidation, TCA cycle, oxidative phosphorylation (OXPHOS) and other bioenergetic processes were coordinately up-regulated, indicating that liver cells confront T2D by increasing energy expenditure and activating pathways that rid themselves of the constitutively increased flux of glucose and lipid. Notably, activation of OXPHOS is immediately related to the overproduction of reactive oxygen species (ROS), which causes oxidative stress within the cells. Increased oxidative stress was also evidenced by our post-translational modification profiles, such that mitochondrial proteins were heavier hydroxylated during T2D development. Moreover, we observed a distinct depression of anti-apoptosis and anti-oxidative stress proteins, which might reflect higher apoptotic index under diabetic stage. We suggest that such changes in systematic metabolism are causally linked to the development of T2D. Comparing proteomics data against microarray data, we demonstrated that many T2D-related alterations were unidentifiable by either proteomic or genomic approaches alone, underscoring the importance of integrating different approaches. Our compendium could help to unveil pathogenic events in mitochondria leading to T2D, and be useful for the discovery of diagnosis biomarker and therapeutic targets of T2D.