Reactive oxygen species (ROS) production has been associated with neuronal death. ROS are also involved in mitochondrial fission, which is mediated by Dynamin-related protein 1 (Drp1). The regulation of mitochondrial fragmentation mediated by Drp1 and its relationship to mitochondrial ROS (mtROS) in neuronal death have not been completely clarified. The aim of this study is to evaluate the role of mtROS in cell death and their involvement in the activation of Drp1 and mitochondrial fission in a model of cell death of cultured cerebellar granule neurons (CGN). Neuronal death of CGN induced by potassium deprivation (K5) and staurosporine (ST) triggers mitochondrial ROS production and mitochondrial fragmentation. K5 condition evoked an increase of Drp1 phosphorylation at Ser616, but ST treatment led to a decrease of Drp1 phosphorylation. Moreover, the death of CGN induced by both K5 and ST was markedly reduced in the presence of MitoTEMPO; however, mitochondrial morphology was not recovered. Here, we show that the mitochondria are the initial source of ROS involved in the neuronal death of CGN and that mitochondrial fragmentation is a common event in cell death; however, this process is not mediated by Drp1 phosphorylation at Ser616.