Insulin is both a hormone regulating energy metabolism and a growth factor. We and others have shown that physiological doses of insulin initiate complex signals in primary human and mouse β-cells, but the functional significance of insulin's effects on this cell type remains unclear. In the present study, the role of insulin in β-cell apoptosis was examined. Exogenous insulin completely prevented apoptosis induced by serum withdrawal when given at picomolar or low nanomolar concentrations but not at higher concentrations, indicating that physiological concentrations of insulin are antiapoptotic and that insulin signaling is self-limiting in islets. Insulin treatment was associated with the nuclear localization of Pdx1 and the prosurvival effects of insulin were largely absent in islets 50% deficient in Pdx1, providing direct evidence that Pdx1 is a signaling target of insulin. Physiological levels of insulin did not increase Akt phosphorylation, and the protective effects of insulin were only partially altered in islets lacking 80% of normal Akt activity, suggesting the presence of additional insulin-regulated antiapoptotic pathways. Proteomic analysis of insulin-treated human islets revealed significant changes in multiple proteins. Bridge-1, a Pdx1-binding partner and regulator of β-cell survival, was increased significantly at low insulin doses. Together, these data suggest that insulin can act as a master regulator of islet survival by regulating Pdx1.