Objective To investigate effects of serum of rats with LPS-induced acute lung injury on endothelial cell permeability and the protective effects of Ulinastatin. Methods Fifteen healthy male SD rats were divided into 3 groups, namely group A with nothing as the control group, group B with LPS-induced acute lung injury, group C with LPS-induced acute lung injury plus ulinastatin treatment. Rat pulmonary microvascular endothelial cell (RPMVEC) was isolated and culture from SD rat in vitro. The serum was collected from rats and added into RPMVEC. The effects of serum on monolayer permeability of RPMVEC were observed with trans-well, and F-actin expression was evaluated by flow cytometry. Results Serum of group B induced the increased permeability of RPMVEC monolayer, depolymerization of F-actin. Pretreatment with ulinastatin could lessen these changes. The percentage in change of permeability coefficient(△Pa%) after stimulation with the serum of rats in group A,B and C was （7.31±0.27）%，（30.13±1.24）%，（18.94±0.59）%,respectively, showing statistically significant differences (P<0.05). And the fluorescent numbers of F-actin were 12.52±2.81、1344.26±12.78、654.63±48.28、321.32±32.14, showing statistically significant differences (P<0.05).Conclusions The pro-inflammatory mediators in the serum of rats with LPS-induced acute lung injury increases RPMVEC permeability ,and pretreatment with ulinastatin can lessen the hyperpermeability by inhibiting multiple pro-inflamatory mediators.