The prevalence of diabetes mellitus is increasing dramatically throughout the world, and the disease has become a major public health issue. The most common form of the disease, type 2 diabetes, is characterized by insulin resistance and insufficient insulin production from the pancreatic beta-cell. Since glucose is the most potent regulator of beta-cell function under physiological conditions, identification of the insulin secretory defect underlying type 2 diabetes requires a better understanding of glucose regulation of human beta-cell function. To this aim, a bottom-up LC-MS/MS-based proteomics approach was used to profile pooled islets from multiple donors under basal (5 mM) or high (15 mM) glucose conditions. Our analysis discovered 256 differentially abundant proteins (∼p < 0.05) after 24 h of high glucose exposure from more than 4500 identified in total. Several novel glucose-regulated proteins were elevated under high glucose conditions, including regulators of mRNA splicing (pleiotropic regulator 1), processing (retinoblastoma binding protein 6), and function (nuclear RNA export factor 1), in addition to neuron navigator 1 and plasminogen activator inhibitor 1. Proteins whose abundances markedly decreased during incubation at 15 mM glucose included Bax inhibitor 1 and synaptotagmin-17. Up-regulation of dicer 1 and SLC27A2 and down-regulation of phospholipase Cβ4 were confirmed by Western blots. Many proteins found to be differentially abundant after high glucose stimulation are annotated as uncharacterized or hypothetical. These findings expand our knowledge of glucose regulation of the human islet proteome and suggest many hitherto unknown responses to glucose that require additional studies to explore novel functional roles.