Type 2 diabetes (T2D) arises when pancreatic beta-cells fail to compensate for systemic insulin resistance with appropriate insulin secretion. However, the link between insulin resistance and beta-cell failure in T2D is not fully understood. To explore this association, we studied transgenic MKR mice that initially develop insulin resistance in skeletal muscle, but by 8 weeks of age have T2D. In the present study, global islet protein and gene expression changes were characterized in diabetic MKR versus non-diabetic control mice at 10 weeks of age. Using a quantitative proteomic approach (iTRAQ), 159 proteins were differentially expressed in MKR compared to control islets. Marked up-regulation of protein biosynthesis and ER stress pathways and parallel down-regulation in insulin processing/secretion, energy utilization and metabolism were observed. A fraction of the differentially expressed proteins identified (including Glut2, Dnajc3, Vamp2, Rab3a and PC1/3) were previously linked to insulin-secretory defects and T2D. However, many proteins for the first time were associated with islet dysfunction; including the unfolded protein response proteins (Erp72, Erp44, Erp29, Ppib, Fkbp2, Fkbp11 and Dnajb11), ER associated degradation proteins (Vcp and Ufm-1), and multiple proteins associated with mitochondrial energy metabolism (Ndufa9, Uqcrh, Cox2, Cox4i1, Cox5a, Atp6v1b2, Atp6v1h, Ant1, Ant2, Etfa and Etfb). The mRNA expression level corresponding to these proteins was examined by microarray and then a small subset was validated using qPCR and western blot analyses. Importantly, ~54% of differentially expressed proteins in MKR islets (including proteins involved in proinsulin processing, protein biosynthesis and mitochondrial oxidation) showed changes in the proteome but not transcriptome, suggesting post-transcriptional regulation. These results underscore the importance of integrated mRNA and protein expression measurements and validate the use of the iTRAQ method combined with microarray to assess global protein and gene changes involved in the development of T2D.