Insulin is believed to regulate glucose homeostasis mainly via direct effects on the liver, muscle, and adipose tissues. The contribution of insulin's central nervous system effects to disorders of glucose metabolism has received less attention. To evaluate whether postnatal reduction of insulin receptors (IRs) within the ventromedial hypothalamus (VMH), a brain region critical for glucose sensing, contributes to disorders of peripheral glucose metabolism, we microinjected a lentiviral vector expressing an antisense sequence to knockdown IRs or a control lentiviral vector into the VMH of nonobese nondiabetic rats. After 3-4 mo, we assessed 1) glucose tolerance, 2) hepatic insulin sensitivity, and 3) insulin and glucagon secretion, using the glucose clamp technique. Knockdown of IRs locally in the VMH caused glucose intolerance without altering body weight. Increments of plasma insulin during a euglycemic clamp study failed to suppress endogenous glucose production and produced a paradoxical rise in plasma glucagon in the VMH-IR knockdown rats. Unexpectedly, these animals also displayed a 40% reduction (P < 0.05) in insulin secretion in response to an identical hyperglycemic stimulus (∼220 mg/dl). Our data demonstrate that chronic suppression of VMH-IR gene expression is sufficient to impair glucose metabolism as well as α-cell and β-cell function in nondiabetic, nonobese rats. These data suggest that insulin resistance within the VMH may be a significant contributor to the development of type 2 diabetes.