The post-translational modifications of Ser and Thr residues by O-linked β-N-acetylglucosamine (O-GlcNAc), ie, O-GlcNAcylation, is considered a key means of regulating signaling, in a manner analogous to protein phosphorylation. Furthermore, it has been suggested that the increased flux of glucose through the hexosamine biosynthetic pathway (HBP) stimulates O-GlcNAcylation, and that this may be responsible for many of the manifestations of type 2 diabetes mellitus. To determine whether excessive O-GlcNAcylation of target proteins results in pancreatic β cell dysfunction, we increased nucleocytoplasmic protein O-GlcNAcylation levels in β cells by exposing them to streptozotocin and/or glucosamine. Streptozotocin and glucosamine co-treatment increased O-GlcNAcylated proteomic patterns as assessed by immunoblotting, and these increases in nuclear and cytoplasmic protein O-GlcNAcylations were accompanied by impaired insulin secretion and enhanced apoptosis in pancreatic β cells. This observed β cell dysfunction prompted us to examine Akt and Bcl-2 family member proteins to determine which proteins are O-GlcNAcylated under conditions of high HBP throughput, and how these proteins are associated with β cell apoptosis. Eventually, we identified ten new O-GlcNAcylated proteins that were expressed during β cell apoptosis, and analyzed the functional implications of these proteins in relation to pancreatic β cell dysfunction.