Abstract: Objective To investigate the effect of Ulinastatin on inhibiting the apoptosis of rat bone marrow-derived mesenchymal stem cells(BMSC) induced by ischemia. Methods Twenty-five SPF grade C57BL/6J male rats were randomly divided into normal control group, ischemic model group, Ulinastatin group, Ulinastatin + AKT pathway inhibitor group, Ulinastatin + ERK pathway inhibitor group, and normal control group without any pretreatment. Ischemic model group only treated with ischemia; Ulinastatin group, Ulinastatin + AKT pathway inhibitor (LY294002) group and Ulinastatin + ERK pathway inhibitor (PD98059) group were pretreated with Ulinastatin, Ulinastatin+LY294002 and ulinastatin+PD98059 respectively for 1 hour before ischemic treatment of BMSC. Ulinastatin concentration was 500 U/ml. Results After 6 hours of culture, the percentage of apoptosis was significantly higher than that at 3 hours of culture, and there was no significant difference between the other adjacent time points. There was significant difference between the apoptosis rate of normal control group and ischemic apoptosis model group. Caspase-3 of ischemic group was significantly higher than that of normal control group. Caspase-3 levels in rats treated with Ulinastatin + AKT pathway inhibitor and ulinastatin + ERK pathway inhibitor were significantly higher than those in rats treated with Ulinastatin, and caspase-3 levels in rats treated with Ulinastatin + AKT pathway inhibitor were significantly lower than those in rats treated with Ulinastatin + ERK pathway inhibitor (t=3.289, P=0.046). Conclusion The culture of 6 h is the most efficient and suitable for the best ischemia time. Ulinastatin can effectively inhibit the apoptosis of rat BMSC. Ulinastatin is more effective by ERK pathway than the AKT inhibitory pathway.