Neurons contain a high number of mitochondria, these neuronal cells produce elevated levels of oxidative stress and live for a long time without proliferation; therefore, mitochondrial homeostasis is crucial to their health. Investigations have recently focused on mitochondrial dynamics revealing the ability of these organelles to change their distribution and morphology. It is known that mitochondrial fission is necessary for the transmission of mitochondria to daughter cells during mitosis and mitochondrial fragmentation has been used as an indicator of cell death and mitochondrial dysfunction. Oxidative stress is a trigger able to induce changes in the mitochondrial network. The aim of the present study was to determine the effects of melatonin on the mitochondrial network in HT22 serum-deprived cells. Our results showed that serum deprivation increased reactive oxygen species (ROS) content, promoted the activation of plasma membrane voltage-dependent anion channels (VDACs) and affected the expression of pDRP1 and DRP1 fission proteins. Moreover, parallel increases in apoptotic and autophagic features were found. Damaged and dysfunctional mitochondria are deleterious to the cell; hence, the degradation of such mitochondria through mitophagy is crucial to cell survival. Our results suggest that melatonin supplementation reduces cell death and restores mitochondrial function through the regulation of autophagy.