Recent evidence suggests that insulin signaling through the insulin receptor A type (Ex11-), regulates insulin gene transcription. Because chronic hyperglycemia negatively affects insulin receptor function and regulates alternative splicing of the insulin receptor, we inquired whether chronic exposure of pancreatic beta-cells to high glucose results in alterations in insulin signaling due to changes in insulin receptor expression and relative abundance of its spliced isoforms. Our results demonstrate that the insulin receptor is localized in insulin secretory vescicles in human pancreatic beta-cells. Furthermore, we find that alterations in insulin expression and secretion caused by chronic exposure to high glucose are paralleled by decreased insulin receptor expression and increased relative abundance of the Ex11+ isoform in both human islets and RIN beta-cells. PDX-1 and HMGI(Y) transcription factors are down-regulated by high glucose. These changes are associated with defects in insulin signaling involving insulin receptor-associated PI 3-kinase/Akt/PHAS-I pathway in RIN beta-cells. Re-expression in RIN beta-cells chronically exposed to high glucose of the Ex11-, but not the Ex11+, isoform restored insulin mRNA expression. These data suggest that changes in early steps of insulin receptor signaling may play a role in determining beta-cell dysfunction caused by chronic hyperglycemia.