Objective The study aimed to explore the relationship between AIF related pathway and the inju-ring of cultured SGNs (spiral ganglion neurons)by glutamate toxicity,and to find AIF expression and distribution changes in SGNs.Methods SGNs of 40 newborn rats within 3 day were obtained and cultured in vitro.Cultured cells were divided into four groups:the normal control group,10 mM,20 mM and 40 mM glutamate injured group, separately.After 48 h hours culturing,optical microscopy,immune fluorescence staining and real-time fluores-cence quantitative PCR were used to observe the morphology,AIF distribution,and AIF,calpain,Caspase3 expres-sion changes in SGNs in vitro.TUNEL was used to verify the cell apoptosis.ResuIts Noticeable morphological chan-ges and cell apoptosis were occurred in 20 mM glutamate group,with AIF nuclear translocation.AIF gene expression was significantly higher than normal after glutamate administration (P<0.05);moreover,calpain gene expression increased(P<0.05);but caspase3 expression was not statistically significantly increased in all glutamate treated groups (P>0.05). ConcIusion In the process of cultured SGNs injured by glutamate,AIF participated in the cell apoptosis.Noticeable cell apoptosis were occurred in 20 mM glutamate group with AIF nuclear translocation.Calpain up-expression also contributed to excitatory neurotransmitter injury on SGNs,but Caspase 3 had no obvious effects.