We previously generated cytochrome P450 4F2 (CYP4F2) transgenic mice that have high levels of 20-hydroxyeicosatetraenoic acid (20-HETE) production; these mice exhibit both hypertension and hyperglycemia without insulin resistance. Currently, it is unclear whether and how 20-HETE affects insulin secretion, thus resulting in hyperglycemia. In this study, we found that 20-HETE attenuated glucose-stimulated insulin secretion (GSIS) in CYP4F2 transgenic mice as well as in rat insulinoma INS-1E cells treated with 0.5 muM 20-HETE. HET0016, a selective inhibitor of 20-HETE synthesis, reversed the reduction in GSIS leading to a decrease in blood glucose in the transgenic mice. Furthermore, the expression of glucose transporter 2 (Glut2), Ser473 phosphorylation of protein kinase B (AKT), and Ser9 phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) were decreased in CYP4F2 transgenic mice compared with wild-type mice. In vitro experiments in INS-1E cells revealed that 20-HETE activated the AKT/GSK-3beta pathway and thereby decreased Glut2 expression by inhibiting activator protein 1 (AP-1). TWS119, a GSK-3beta selective inhibitor, blocked the 20-HETE-mediated reduction in Glut2 expression. Therefore, we concluded that 20-HETE inhibition of Glut2 contributes to the reduction in GSIS, at least in part, through the AKT/GSK-3beta/AP-1/Glut2 pathway.