It has been proposed that endogenous hexokinases of the pancreatic beta cell control the rate of glucose-stimulated insulin secretion and that genetic defects that reduce beta-cell hexokinase activity may lead to diabetes. To test these hypotheses, we have produced transgenic mice that have a 2-fold increase in hexokinase activity specific to the pancreatic beta cell. This increase was sufficient to significantly augment glucose-stimulated insulin secretion of isolated pancreatic islets, increase serum insulin levels in vivo, and lower the blood glucose levels of transgenic mice by 20-50% below control levels. Elevation of hexokinase activity also significantly reduced blood glucose levels of diabetic mice. These results confirm the role of beta-cell hexokinase activity in the regulation of insulin secretion and glucose homeostasis. They also provide strong support for the proposal that reductions in beta-cell hexokinase activity can produce diabetes.