Abstract: Objective To investigate the effect of ulinastatin pretreatment on isoflurane-induced mitochondria-dependent neuronal apoptosis in the hippocampus of rats. Methods Thirty-six male SD rats were randomly assigned into control group, isoflurane group and ulinastatin group. In the latter two groups, the rats were subjected to acute exposure to 0.75% isoflurane for 6 h and pretreated with 50 000 U/kg of ulinastatin before isoflurane exposure, respectively. After the treatments, apoptosis of the hippocampal neurons was detected using TUNEL assay, and the mitochondrial membrane potential ( △ ψm) was measured using JC-1 mitochondrial membrane potential kit; cytochrome C release and caspase-3 activity were examined with Western blotting, and intracellular reactive oxygen species (ROS) was detected using the fluorescent probe H2DCFDA. Results Compared with those in the control group, the rats with acute exposure to isoflurane showed markedly increased TUNELpositive cells in the hippocampus (P<0.05), which were obviously reduced by ulinastatin pretreatment (P<0.05). The △ψm of the hippocampal neurons was significantly reduced after isoflurane exposure (P<0.05), and was partly recovered by ulinastatin pretreatment (P<0.05). Acute exposure to isoflurane resulted in obviously increased cellular ROS, cytochrome C release and caspase-3 activity in the hippocampal neurons (P<0.05), and these changes were significantly inhibited by ulinastatin pretreatment (P<0.05). Conclusion Ulinastatin pretreatment provides neuroprotection against isoflurane-induced apoptosis of the hippocampal neurons in rats possibly by inhibiting mitochondria-dependent apoptosis pathway.