Fission and fusion of mitochondrial tubules are the major processes regulating mitochondrial morphology. However, the physiological significance of mitochondrial shape change is poorly understood. Glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells requires mitochondrial ATP production which evokes Ca2+ influx through plasma membrane depolarization, triggering insulin vesicle exocytosis. Therefore, GSIS reflects mitochondrial function and can be used for evaluating functional changes associated with morphological alterations of mitochondria. Using the insulin-secreting cell line INS-1E, we found that glucose stimulation induced rapid mitochondrial shortening and recovery. Inhibition of mitochondrial fission through expression of the dominant-negative mutant DLP1-K38A eliminated this dynamic mitochondrial shape change and, importantly, blocked GSIS. We found that abolishing mitochondrial morphology change in glucose stimulation increased the mitochondrial inner membrane proton leak, and thus significantly diminished the mitochondrial ATP producing capacity in response to glucose stimulation. These results demonstrate that dynamic change of mitochondrial morphology is a previously unrecognized component for metabolism-secretion coupling of pancreatic β-cells by participating in efficient ATP production in response to elevated glucose levels.