Abstract This is the first systematic study on the pancreatic β cell function in the heterozygous mouse with targeted disruption of the β cell glucokinase gene. The hetetrozygotes’ β cell displayed the following characteristics: (1) impaired glucose sensitivity with normal glucose responsiveness, (2) poor discrimination of α and β glucose anomers, and (3) normal response to glucose in the presence of 25 mM K+and 150 μM diazoxide. Both the first and the second phases of glucose-stimulated insulin release were depressed. Although the heterozygotes were mildly hyperglycemic, insulin treatment further suppressed β cell function, implying the β cell glucose toxicity is not the cause of impaired glucose sensitivity. The data are compatible with the glucokinase glucose sensor concept inasmuch as glucose sensitivity is reduced in the heterozygotes’ β cell. The anomeric malaise and preservation of the ATP-sensitive K+channel-independent glucose action were considered due to chronic hyperglycemia.