Objective Emulsified isoflurane (8% ,vol/vol) is a kind of lipid based formation for intravenous administration. The purpose of this study is to evaluate whether emulsified isoflurane induces hemolysis or not in vitro and in vivo. Methods In hemolysis test in vitro, a male rabbit was used to prepare 2% (vol/vol) erythrocyte suspensions for measuring degree of hemolysis of emulsified isoflurane at the doses from 12 to 0. 3 g/L. In hemolysis test in vivo,4 male Beagle dogs were intravenously adminstrated emulsified isoflurane 225.6 mg/kg in 3-5 min. 5 ml samples of venous blood were collected from each dog at 0 min (start of injection) ,5 min,30 min,1 h,2 h,4 h,8 h,1 d,2 d,3 d and 6 d after the adminstration of emulsified isoflurance for measuring erythrocyte morphology,reticulocyte counts, the concentrations of free hemoglobin,haptoglobin,and bilirubin. At the same time, urinary blood, urinary bilirubin and urobilinogen were also measured before and after adminstration. Results In vitro experiment,emulsified isoflurane led to hemolysis at the concentrations from 12 to 1.2 g/L. However,no hemolysis was found at the concentrations from 0. 6 to 0. 3 g/L. In vivo experiment,with the exception of a slight reduction in indirect bilirubin and a mild increase in direct bilirubin at 5 min (P < 0.05 ) , others such as total bilirubin, retculocyte counts, haptoglobin, free hemoglobin,urinary bilirubin,urinary blood, and urinary urobinogen were not significantly different compared with their corresponding values before injection. There was also no significant difference in erythrocyte fragmentations at 0 min,5 min,30 min,1 h,2 h,4 h,8 h,1 d,2 d,3 d and 6 d after injection of emusified isoflurane, and none of macrocytes and nucleated red cells was noted on all blood films. Conclusions Emulsified isoflurane at concentrations recommended for clinical trials did not cause hemolysis in vitro and in vivo.