A large number of studies have demonstrated that inhalation anesthetic isoflurane induced neural cell death by apoptosis in various cell and animal models. Emulsified isoflurane (EIso) is a new type of intravenous preparation of isoflurane that attracts increasing research attention as a promising clinical agent due to its both advantages as an intravenous and inhalation anesthetics medication. However, its safety and underlying molecular mechanism of neurotoxicity largely remain unknown. Therefore, it is meaningful to investigate the safety of EIso and to further elucidate its mechanism of anesthetic neurotoxicity. Human neuroblastoma SHSY-5Y cells were cultured, followed by a random exposure to one of three doses of EIso (0.56mmol/l, 1.12mmol/l, and 2.24mmol/l) or the corresponding intralipid as vehicle (0.3956μl/ml, 0.7912μl/ml and 1.5824μl/ml) for 6h, 12h or 24h. Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assay and the morphological changes were determined by a light microscope. We detect JNK, p-JNK and cytochrome C (cyto C) protein levels by western blotting. SP600125, a specific inhibitor of JNK, was used to detect the role of JNK pathway in the neurotoxicity of EIso. Our study showed that EIso reduced the viability of SHSY-5Y cells in a dose- and time-dependent manner. 0.56mmol/l EIso has no significant effects on cell viability, while 1.12mmol/l of EIso with 24-h and 2.24mmol/l of EIso with over 12-h exposure notably reduced cell viability. EIso dramatically increased the levels of p-JNK and cyto C. The JNK pathway inhibitor SP600125 significantly increased the cell viability of SHSY-5Y cells induced by EIso. These findings suggest that EIso induces damage in human neuroblastoma SHSY-5Y cells by JNK signaling pathway activation and cyto C release. SP600125 protects neural cells against EIso-induced injury. Our findings provide a new insight in the exploration of potential novel therapeutic strategies for the treatment of EIso-induced neurotoxicity and other neurodegenerative diseases.