A relative decrease in -cell mass is key in the pathogenesis of type 1 diabetes, type 2 diabetes and in the failure of transplanted islet grafts. It is now clear that -cell duplication plays a dominant role in the regulation of adult -cell mass. Knowledge of the endogenous regulators of -cell replication is therefore critical for understanding the physiological control of -cell mass and for harnessing this process therapeutically. We have shown that concentrations of insulin known to exist in vivo act directly on -cells to promote survival. Whether insulin stimulates adult -cell proliferation remains unclear. We tested this hypothesis using dispersed primary mouse islet cells double-labeled with BrdU and insulin antisera. Treating cells with 200 pM insulin significantly increased proliferation from a baseline rate of 0.15% per day. Elevating glucose from 5 mM to 15 mM did not significantly increase -cell replication. -cell proliferation was inhibited by somatostatin, as well as inhibitors of insulin signaling. Interestingly, inhibiting Raf-1 kinase blocked proliferation stimulated by low, but not high (super-physiological) insulin doses. Insulin-stimulated MIN6 cell proliferation was dependent on both PI3-kinase/Akt and Raf-1/Mek pathways. Over-expression of Raf-1 was sufficient to increase proliferation in the absence of insulin, whereas a dominant-negative Raf-1 reduced proliferation in the presence of 200 pM insulin. Together, these results demonstrate for the first time that insulin, at levels that have been measured in vivo, can directly stimulate -cell proliferation and that Raf-1 kinase is involved in this process. These findings have significant implications for the understanding of the regulation of -cell mass in both the hyperinsulinemic and insulin-deficient states that occur in the various forms of diabetes.