Chronic abuse of psychoactive drugs including cocaine causes addiction, dependence, and brain disorders through the alteration of neuronal plasticity. Mitochondrial fission and fusion, collectively called as mitochondrial dynamics, participates in not only the maintenance of neuronal homeostasis but also reorganization of neuronal circuits. The purpose of this study is to examine effects of direct and repeated exposure of cocaine on mitochondrial dynamics in neuronal cells in vitro. Repeated exposure to 600 µM cocaine (2∼3 times per week) of Neuro2a mouse neuroblastoma cells for 3 weeks resulted in decrease of mitochondrial transmembrane potential, activation of autophagy, and upregulation of Parkin, a protein involved in mitochondrial autophagy. Increased expression of mitochondrial fission genes and significant increase in the ser-616 phosphorylated-DRP1, the key regulator of mitochondrial fission, were observed in the cells exposed repeatedly to 600 µM cocaine. Electron microscopy showed significant increase in the number of mitochondria in cocaine-treated cells compared with control cells. Thus, our results show that repeated cocaine exposure not only causes mitochondrial dysfunction but also alters mitochondrial dynamics in Neuro2a cells. Changes in the mitochondrial dynamics might be involved in neural adaptation during repeated cocaine exposure.