Neurotoxicity remains a poorly characterized an adverse effect associated with colistin therapy. The aim of the present study was to investigate the mechanism of colistin-induced neurotoxicity using the mouse neuroblastoma-2a (N2a) cell line. Colistin induced apoptotic neuronal cell death in a dose dependent manner (colistin at 0-200 μM). Colistin treatment (at 50, 100 and 200 μM) for 24 h, significant increased the reactive oxygen species (ROS) levels; while a concomitant decrease in the activities of SOD and catalase and GSH levels were detected. Mitochondrial dysfunction was evident from the dissipation of membrane potential and the increase of Bax/Bcl-2, followed by the release of cytochrome c. Caspase-3/7, 8 and 9 activation was also detected. The results of qRT-PCR showed that colistin treatment activated the gene expression of p53, bax and caspase-8, significantly increased to 1.6, 3.3 and 2.2 fold (colistin at 200 μM) (all p<0.01), respectively. The formation of autophagic vacuoles was evident with significant increases (all p<0.05 or 0.01) of both of Beclin-1 and LC3B following colistin treatment (50-200 μM), indicated that the cells were undergoing autophagy. In summary, our study reveals that colistin induced neuronal cell death involves ROS mediated oxidative stress and mitochondrial dysfunction, followed by caspase-dependent apoptosis and autophagy. This knowledge-base of the neuronal signaling pathways of colistin-induced neurotoxicity will greatly facilitate the discovery of neuro-protective agents for use in combination with colistin to prevent this undesirable side-effect.