Insulin and insulin-like growth factor I (IGF-I) are ubiquitous hormones that regulate growth and metabolism of most mammalian cells, including pancreatic beta-cells. In addition to being an insulin secretagogue, glucose regulates proliferation and survival of beta-cells. However, it is unclear whether the latter effects of glucose occur secondary to autocrine activation of insulin signaling proteins by secreted insulin. To examine this possibility we studied the effects of exogenous glucose or insulin in beta-cell lines completely lacking either insulin receptors (betaIRKO) or insulin receptor substrate 2 (betaIRS2KO). Exogenous addition of either insulin or glucose activated proteins in the insulin signaling pathway in control beta-cell lines with the effects of insulin peaking earlier than glucose. Insulin stimulation of betaIRKO and betaIRS2KO cells led to blunted activation of phosphatidylinositol 3-kinase and Akt kinase, while surprisingly, glucose failed to activate either kinase but phosphorylated extracellular signal-regulated kinase. Control beta-cells exhibited low expression of IGF-1 receptors compared to compensatory upregulation in betaIRKO cells. The signaling data support the slow growth and reduced DNA and protein synthesis in betaIRKO and betaIRS2KO cells in response to glucose stimulation. Together, these studies provide compelling evidence that the growth and survival effects of glucose on beta-cells require activation of proteins in the insulin signaling pathway.