The present study identified the cytotoxic effects of etomidate on the N2a neuroblastoma cell line. Etomidate induced apoptosis in N2a cells in a concentration‑dependent manner, which was confirmed by western blotting and flow cytometry. Phase contrast microscopy was used to analyze the effect of etomidate on morphological characteristics. The number of the apoptotic cells was increased and confirmed by DAPI and PI staining, which served as a characteristic hallmark of apoptosis. Additionally, etomidate led to loss of mitochondrial membrane potential and resulted in the generation of reactive oxygen species in N2a cells. The western blot analysis revealed that N2a cells treated with etomidate had a significant modulation of pro‑apoptotic proteins, includingpoly ADP‑ribose polymerase (PARP), cleaved PARP, caspase‑9 and procaspase‑3. In conclusion, the present study determined that etomidate induced cytotoxic and apoptotic effects in N2a brain tumor cells in vitro.