Background Increased uncoupling protein-2 (UCP-2) expression has been associated with impaired insulin secretion, whereas UCP-3 protein levels are decreased in the skeleton muscle of type-2 diabetic subjects. In the present studies we hypothesize an opposing effect of glucose on the regulation of UCP-2 and UCP-3 in pancreatic islets. Methodology Dominant negative UCP-2 and wild type UCP-3 adenoviruses were generated, and insulin release by transduced human islets was measured. UCP-2 and UCP-3 mRNA levels were determined using quantitative PCR. UCP-2 and UCP-3 protein expression was investigated in human islets cultured in the presence of different glucose concentrations. Human pancreatic sections were analyzed for subcellular localization of UCP-3 using immunohistochemistry. Principal Findings Dominant negative UCP-2 expression in human islets increased insulin secretion compared to control islets (p<0.05). UCP-3 mRNA is expressed in human islets, but the relative abundance of UCP-2 mRNA was 8.1-fold higher (p<0.05). Immunohistochemical analysis confirmed co-localization of UCP-3 protein with mitochondria in human beta-cells. UCP-2 protein expression in human islets was increased ∼2-fold after high glucose exposure, whereas UCP-3 protein expression was decreased by ∼40% (p<0.05). UCP-3 overexpression improved glucose-stimulated insulin secretion. Conclusions UCP-2 and UCP-3 may have distinct roles in regulating beta-cell function. Increased expression of UCP-2 and decreased expression of UCP-3 in humans with chronic hyperglycemia may contribute to impaired glucose-stimulated insulin secretion. These data imply that mechanisms that suppress UCP-2 or mechanisms that increase UCP-3 expression and/or function are potential therapeutic targets to offset defects of insulin secretion in humans with type-2 diabetes.