Although many studies using rodent islets and insulinoma cell lines have been performed to determine the role of insulin in the regulation of islet function, the autocrine effect of insulin on insulin gene expression is still controversial, and no consensus has yet been achieved. Because very little is known about the insulin signaling pathway in human islets, we used single-cell RT-PCR to profile the expression of genes potentially involved in the insulin signaling cascade in human β-cells. The detection of mRNAs for insulin receptor (IR)_A and IR_B; insulin receptor substrate (IRS)-1 and IRS-2; phosphoinositide 3-kinase (PI3K) catalytic subunits p110α, p110β, PI3KC2α, and PI3KC2γ; phosphoinositide-dependent protein kinase-1; protein kinase B (PKB)α, PKBβ, and PKBγ in the β-cell population suggests the presence of a functional insulin signaling cascade in human β-cells. Small interfering RNA–induced reductions in IR expression in human islets completely suppressed glucose-stimulated insulin gene expression, suggesting that insulin regulates its own gene expression in human β-cells. Defects in this regulation may accentuate the metabolic dysfunction associated with type 2 diabetes.