The study investigated the effect of colistin exposure on calcium homeostasis and mitochondria functions in neurons. We used an in vitro drug model to induce neurotoxicity that closely mimic the in vivo condition by exposing primary cultures of chick cortex neurons to different concentrations of 0, 0.83, 4.15 and 8.3?g/mL colistin. The cell activity was determined by methods of MTT and lactate dehydrogenase release at 24h post-beginning. The membrane potential (??m) and ultrastructure of mitochondrial were assessed. The calcium ion concentration within neurons ([Ca2+]i) was detected using the Fura3/AM as the probe and expression level of intracellular calmodulin (CaM) mRNA was detected by reverse transcription polymerase chain reaction. The results showed that, in the 4.15 and 8.3?g/mL colistin groups, the functions of mitochondria altered, the ??m was significantly decreased and the mitochondrial cristae was swollen and even vacuolar degeneration in mitochondria occurred. Moreover, the expression level of colistin could decrease CaM mRNA, and increase free calcium concentration. The present work revealed that colistin-induced mitochondria dysfunction and calcium homeostasis disequilibrium, providing new insight into the toxicological mechanism of colistin in neurons.