Persistent hyperglycemia is causally associated with pancreatic β-cell dysfunction and loss of pancreatic insulin. Glucose normally enhances β-cell excitability through inhibition of K ATP channels, opening of voltage-dependent calcium channels, increased [Ca 2+ ] i , which triggers insulin secretion. Glucose-dependent excitability is lost in islets from K ATP -knockout (K ATP -KO) mice, in which β-cells are permanently hyperexcited, [Ca 2+ ] i, is chronically elevated and insulin is constantly secreted. Mouse models of human neonatal diabetes in which K ATP gain-of-function mutations are expressed in β-cells (K ATP -GOF) also lose the link between glucose metabolism and excitation-induced insulin secretion, but in this case K ATP -GOF β-cells are chronically underexcited, with permanently low [Ca 2+ ] i and lack of glucose-dependent insulin secretion. We used K ATP -GOF and K ATP -KO islets to examine the role of altered-excitability in glucotoxicity. Wild-type islets showed rapid loss of insulin content when chronically incubated in high-glucose, an effect that was reversed by subsequently switching to low glucose media. In contrast, hyperexcitable K ATP -KO islets lost insulin content in both low- and high-glucose, while underexcitable K ATP -GOF islets maintained insulin content in both conditions. Loss of insulin content in chronic excitability was replicated by pharmacological inhibition of K ATP by glibenclamide, The effects of hyperexcitable and underexcitable islets on glucotoxicity observed in in vivo animal models are directly opposite to the effects observed in vitro: we clearly demonstrate here that in vitro, hyperexcitability is detrimental to islets whereas underexcitability is protective.