An important goal in diabetes research is to understand the processes that trigger endogenous beta-cell proliferation. Hyperglycemia induces beta-cell replication, but the mechanism remains debated. A prime candidate is insulin, which acts locally through the insulin receptor. Having previously developed an in vivo mouse hyperglycemia model, we tested whether glucose induces beta-cell proliferation through insulin signaling. By using mice lacking insulin signaling intermediate insulin receptor substrate 2 (IRS2), we confirmed that hyperglycemia-induced beta-cell proliferation requires IRS2 both in vivo and ex vivo. Of note, insulin receptor activation was not required for glucose-induced proliferation, and insulin itself was not sufficient to drive replication. Glucose and insulin caused similar acute signaling in mouse islets, but chronic signaling differed markedly, with mammalian target of rapamycin (MTOR) and extracellular signal-related kinase (ERK) activation by glucose and AKT activation by insulin. MTOR but not ERK activation was required for glucose-induced proliferation. Cyclin D2 was necessary for glucose-induced beta-cell proliferation. Cyclin D2 expression was reduced when either IRS2 or MTOR signaling was lost, and restoring cyclin D2 expression rescued the proliferation defect. Human islets shared many of these regulatory pathways. Taken together, these results support a model in which IRS2, MTOR, and cyclin D2, but not the insulin receptor, mediate glucose-induced proliferation.