Chronic hyperglycaemia impairs insulin secretion by disrupting beta-cell metabolism and reducing expression of insulin. These changes are mediated by glucose metabolites downstream of glucokinase. We hypothesized that chronically enhancing glucokinase activity at low glucose would mimic the effects of glucotoxicity and, conversely, that reducing glucokinase activity would prevent the deleterious effects of chronic hyperglycaemia. Human islet microtissues or INS-1 cells were cultured at low or high glucose for 2 weeks (islets) or 48 h (INS-1 cells) in the presence or absence of either a glucokinase activator (MK-0941 or dorzagliatin) or a glucokinase inhibitor (mannoheptulose). We measured changes in glucose-stimulated insulin secretion, insulin content, glycogen content, gene expression, and oxygen consumption. Chronic glucokinase activation in low-glucose-cultured islet microtissues and INS-1 cells downregulated insulin secretion and content, impaired mitochondrial metabolism, and altered metabolic gene expression, to the same extent as high-glucose culture. Conversely, reduction of glucokinase activity in high-glucose-cultured islet microtissues by mannoheptulose prevented the changes produced by chronic hyperglycaemia. Moreover, the effects of chronic hyperglycaemia on insulin secretion and metabolic gene expression (but not insulin content) were largely reversed by subsequent inhibition of glucokinase. Chronic glucokinase activation in euglycaemia thus mimics the effects of chronic hyperglycaemia, whereas partial inhibition of glucokinase in hyperglycaemia restores normal beta-cell function. This explains why, in the long term, glucokinase activators are ineffective at controlling glycaemia in type 2 diabetes, and demonstrates that normalizing glucokinase activity can prevent and reverse chronic glucose toxicity.