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Background and aims: The rapid reversibility of recent type 2 diabetes (T2D) by very low-calorie diet in some patients correlates with a marked improvement of glucose-stimulated insulin secretion (GSIS), emphasizing the role of β-cell dysfunction in the early stages of the disease. In search of human-specific mechanisms of β-cell dysfunction, we extensively characterized the glucotoxic alterations of insulin secretion and upstream coupling events in human pancreatic islets cultured for 1 to 3 weeks at ~5, 8, 10 or 20 mmol/l glucose and acutely stimulated by a stepwise increase in glucose concentration. Materials and methods: Islets from 46 non-diabetic (ND) and 6 type 2 diabetic (T2D) donors were obtained from 5 isolation centers over the last 10 years. The islets were precultured 3-7 days in RPMI containing 5 mmol/l glucose and 10% FBS. They were then cultured for 1-3 weeks in the same medium containing 5.5, 8.5, 10.5 or 20.5 mmol/l glucose before measurements of insulin secretion during culture, islet insulin/DNA content ratio, β-cell apoptosis, cytosolic and mitochondrial thiol redox state, and assessment of dynamic insulin secretion and upstream coupling events during stepwise stimulation with glucose (NAD(P)H autofluorescence, ATP/(ATP+ADP) ratio, electrical activity, cytosolic Ca2+ concentration ([Ca2+]c)). Results: Culture of ND-islets for 1 to 3 weeks at ~8, 10 or 20 vs 5 mmol/l glucose did not increase β-cell apoptosis or oxidative stress but concentration-dependently decreased insulin content and increased the β-cell sensitivity to subsequent stimulation with glucose. The islet glucose responsiveness (max amplitude of GSIS per islet) was larger after culture at 8 or 10 vs 5 mmol/l glucose but was markedly reduced after culture at 20 vs 5 mmol/l glucose. In the latter islets, [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were bell-shaped, with a maximal stimulation at 5 to 10 mmol/l glucose followed by a rapid sustained inhibition above that concentration. This glucotoxic alteration was a robust characteristic of human islets. It resulted from long-term stimulation of glucose metabolism and was fully reversible after culture at 5 mmol/l glucose. Finally, acute activation/inhibition of glucokinase during perifusion of islets cultured at 20 mmol/l glucose indicated that the acute reduction of [Ca2+]c and insulin secretion above 10 mmol/l glucose was due to overstimulation rather than inhibition of glucose metabolism. Similar results were obtained in islets from T2D-donors. Conclusion: Long-term exposure of human islets to mildly elevated glucose concentrations markedly increases their glucose sensitivity and reveals a bell-shaped glucose response curve for changes in [Ca2+]c and insulin secretion. This human-specific glucotoxic alteration may contribute to β-cell dysfunction in T2D independently from a detectable increase in β-cell apoptosis and oxidative stress.