Glucokinase (Gck) functions as a glucose sensor for insulin secretion, and in mice fed standard chow, haploinsufficiency of β cell–specific Gck (Gck+/–) causes impaired insulin secretion to glucose, although the animals have a normal β cell mass. When fed a high-fat (HF) diet, wild-type mice showed marked β cell hyperplasia, whereas Gck+/–mice demonstrated decreased β cell replication and insufficient β cell hyperplasia despite showing a similar degree of insulin resistance. DNA chip analysis revealed decreased insulin receptor substrate 2 (Irs2) expression in HF diet–fed Gck+/–mouse islets compared with wild-type islets. Western blot analyses confirmed upregulated Irs2 expression in the islets of HF diet–fed wild-type mice compared with those fed standard chow and reduced expression in HF diet–fed Gck+/–mice compared with those of HF diet–fed wild-type mice. HF diet–fed Irs2+/–mice failed to show a sufficient increase in β cell mass, and overexpression of Irs2 in β cells of HF diet–fed Gck+/–mice partially prevented diabetes by increasing β cell mass. These results suggest that Gck and Irs2 are critical requirements for β cell hyperplasia to occur in response to HF diet–induced insulin resistance.