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