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 [Ca²⁺]_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, [Ca²⁺]_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 [Ca²⁺]_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.