As the rate-limiting controller of glucose metabolism, glucokinase represents the primary β-cell "glucose sensor." Inactivation of both glucokinase (GK) alleles results in permanent neonatal diabetes; inactivation of a single allele causes maturity-onset diabetes of the young type 2 (MODY-2). Similarly, mice lacking both alleles (GK-/-) exhibit severe neonatal diabetes and die within a week, whereas heterozygous GK +/- mice exhibit markedly impaired glucose tolerance and diabetes, resembling MODY-2. Glucose metabolism increases the cytosolic [ATP]-to-[ADP] ratio, which closes ATP-sensitive K+ channels (KATP channels), leading to membrane depolarization, Ca2+ entry, and insulin exocytosis. Glucokinase insufficiency causes defective KATP channel regulation, which may underlie the impaired secretion. To test this prediction, we crossed mice lacking neuroendocrine glucokinase (nGK +/-) with mice lacking KATP channels (Kir6.2 -/-). Kir6.2 knockout rescues perinatal lethality of nGK -/-, although nGK-/-Kir6.2-/- animals are postnatally diabetic and still die prematurely. nGK+/- animals are diabetic on the Kir6.2+/+ background but only mildly glucose intolerant on the Kir6.2-/- background. In the presence of glutamine, isolated nGK+/- Kir6.2-/- islets show improved insulin secretion compared with nGK+/-Kir6.2+/+. The significant abrogation of nGK-/- and nGK+/- phenotypes in the absence of KATP demonstrate that a major factor in glucokinase deficiency is indeed altered KATP signaling. The results have implications for understanding and therapy of glucokinase-related diabetes.