Methamphetamine (METH) is an addictive drug that can cause toxicity and degeneration in the brain. Several pieces of evidence have demonstrated that METH toxicity results in increases in oxidative stress that regulate an intracellular signaling cascade that leads to cell death. Recently, several studies have emphasized that the overload of cytosolic calcium levels and mitochondrial fission into a small mitochondrial structure is involved in cell death processes. In the present study, we aimed to investigate the effects of METH toxicity on cytosolic calcium overload and mitochondrial fission in neuroblastoma SH-SY5Y cells. Additionally, the protective effect of melatonin against METH-induced toxicity was also investigated. The results of the present study demonstrated that METH significantly decreases cell viability and increases the levels of mitochondrial fission (Fis1 and Drp1) proteins and pro-apoptotic protein, Bax in isolated mitochondria. The levels of Drp1 in the cytosol of METH-treated cells had no significant differences compared to the control untreated cells. METH also significantly increased the cytosolic calcium levels. Melatonin reversed the toxic effects of METH by restoring cell viability and inhibiting the increase in mitochondrial Fis1 levels and the mitochondrial translocation of Drp1 and Bax. Additionally, melatonin was able to reduce the METH-induced increase in cytosolic calcium levels and fragmented mitochondria into small globular structures in SH-SY5Y cells. The results of the present study demonstrate the potential abilities of melatonin to maintain the homeostasis of mitochondrial dynamics and cytosolic calcium levels in METH-induced toxicity in neuronal cells.