Pancreatic beta-cells sense glucose promoting insulin secretion. Glucose sensing requires the sequential stimulation of glycolysis, mitochondrial metabolism and Ca2+ entry. To elucidate how mitochondrial activation in beta-cells contributes to insulin secretion, we have compared the effects of glucose and the mitochondrial substrate methylsuccinate in INS-1E insulin secreting cells, at the respective concentrations they maximally activate mitochondrial respiration. Both substrates induced insulin secretion with distinct respiratory profiles, mitochondrial hyperpolarization, NADH production and ATP/ADP ratios. In contrast to glucose, methylsuccinate failed to induce large [Ca2+] rises and exocytosis proceeded largely independent of mitochondrial ATP synthesis. Both glucose- and methylsuccinate-induced secretion was blocked by diazoxide, indicating that Ca2+ is required for exocytosis. Dynamic assessment of mitochondrial thiols redox state revealed a less marked reduction in response to methylsuccinate compared to glucose. Our results demonstrate that insulin exocytosis can be promoted by two distinct mechanisms depending on mitochondrial ATP synthesis and large Ca2+-transients or independent of mitochondrial ATP synthesis relying on small calcium signals. We propose that the combined effects of Ca2+ and redox reactions can trigger insulin secretion by these two mechanisms.