Proteomics has rapidly become a major field of research in biology and medicine. Its main aim is to obtain a global overview of the expression pattern of proteins and their relationship in any given condition of a biological system. This knowledge is of particular interest to elucidate the pathogenesis of complex disorders, such as diabetes. Separation of proteins by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS) for their identification is perhaps the most widely used proteomic approach. In this review we have focused our attention on studies that have taken advantage of these methodologies to investigate the proteome of pancreatic islets, β-cells and insulinoma cells in different conditions. As β-cells of the pancreatic islets produce and secrete insulin, the main hormone for control of glucose homeostasis, these analyses may help to elucidate the mechanisms regulating insulin secretion and the development of various forms of diabetes, as well as to identify drug targets for therapeutic approaches.