Insulin and insulin-like growth factor I (IGF-I) are ubiquitous hormones that regulate growth and metabolism of most mammalian cells, including pancreatic β-cells. In addition to being an insulin secretagogue, glucose regulates proliferation and survival of β-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 β-cell lines completely lacking either insulin receptors (βIRKO) or insulin receptor substrate 2 (βIRS2KO). Exogenous addition of either insulin or glucose activated proteins in the insulin signaling pathway in control β-cell lines with the effects of insulin peaking earlier than glucose. Insulin stimulation of βIRKO and βIRS2KO 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 β-cells exhibited low expression of IGF-1 receptors compared to compensatory upregulation in βIRKO cells. The signaling data support the slow growth and reduced DNA and protein synthesis in βIRKO and βIRS2KO cells in response to glucose stimulation. Together, these studies provide compelling evidence that the growth and survival effects of glucose on β-cells require activation of proteins in the insulin signaling pathway.