Pancreatic beta-cell dysfunction is a critical component in the pathogenesis of diabetes. Endoplasmic reticulum (ER) stress is one of the factors that induces pancreatic beta-cell dysfunction, but the underlying mechanisms have not been well elucidated. In this study, we report that a mitochondrial fission modulator, dynamin-related protein I (DRP-1), plays an important role in ER stress-induced beta-cell apoptosis. Induction of DRP-1 expression significantly promoted ER stress-induced apoptosis in the DRP-1 WT (DRP-1 wildtype) inducible beta-cell line, but not in the DRP-1 K38A (a dominant negative mutant of DRP-1) inducible beta-cell line. We further demonstrated that the mitochondrial membrane potential decreased, and that cytochrome c release, caspase-3 activation and generation of reactive oxygen species (ROS) were enhanced by induction of DRP-1 WT, but prevented by DRP-1 K38A in pancreatic beta-cells under ER stress conditions. These results indicate that DRP-1 mediates ER stress-induced pancreatic beta-cell apoptosis.