Aims/hypothesis In islets from individuals with type 2 diabetes and in islets exposed to chronic elevated glucose, mitochondrial energy metabolism is impaired. Here, we studied early metabolic changes and mitochondrial adaptations in human beta cells during chronic glucose stress. Methods Respiration and cytosolic ATP changes were measured in human islet cell clusters after culture for 4 days in 11.1 mmol/l glucose. Metabolomics was applied to analyse intracellular metabolite changes as a result of glucose stress conditions. Alterations in beta cell function were followed using insulin secretion assays or cytosolic calcium signalling after expression of the calcium probe YC3.6 specifically in beta cells of islet clusters. Results At early stages of glucose stress, mitochondrial energy metabolism was augmented in contrast to the previously described mitochondrial dysfunction in beta cells from islets of diabetic donors. Following chronic glucose stress, mitochondrial respiration increased (by 52.4%,p < 0.001) and, as a consequence, the cytosolic ATP/ADP ratio in resting human pancreatic islet cells was elevated (by 27.8%,p < 0.05). Because of mitochondrial overactivation in the resting state, nutrient-induced beta cell activation was reduced. In addition, chronic glucose stress caused metabolic adaptations that resulted in the accumulation of intermediates of the glycolytic pathway, the pentose phosphate pathway and the TCA cycle; the most strongly augmented metabolite was glycerol 3-phosphate. The changes in metabolites observed are likely to be due to the inability of mitochondria to cope with continuous nutrient oversupply. To protect beta cells from chronic glucose stress, we inhibited mitochondrial pyruvate transport. Metabolite concentrations were partially normalised and the mitochondrial respiratory response to nutrients was markedly improved. Furthermore, stimulus-secretion coupling as assessed by cytosolic calcium signalling, was restored. Conclusion/interpretation We propose that metabolic changes and associated mitochondrial overactivation are early adaptations to glucose stress, and may reflect what happens as a result of poor blood glucose control. Inhibition of mitochondrial pyruvate transport reduces mitochondrial nutrient overload and allows beta cells to recover from chronic glucose stress.