Objective: Ulinastatin reduces the high permeability of vascular endothelial cells induced by tumor necrosis factor alpha (INF-alpha). This study investigated the molecular mechanism behind this effect, with the aim of understanding the action of ulinastatin in sepsis therapy and exploring novel therapeutic strategies for sepsis patients. Methods: A TNF-alpha treated human umbilical vein endothelial cell line (EA.hy926) was employed as an inflammation model. Horseradish peroxidase permeability assays and an epithelial voltmeter method were used to measure the permeability of EA.hy926 cells. Immunocytochemistry was used to assay the expression of p-MYPT1 and the distribution and morphology of F-actin; the expression of the key molecules related to vascular endothelial permeability. (RhoA, ROCK2, MYPT1, p-MYPT1 and VE-cadherin) was detected by immunocytochemistry assays, western blotting and quantitative real-time polymerase chain reaction. Results: After incubation with TNF-a or septic serum, the transendothelial electrical resistance of EA.hy926 cells decreased and the permeability of the cells increased significantly (all P < 0.05). The expression of p-MYPT1 was higher and VE-cadherin was lower compared with the control group, and F-actin was redistributed, with the formation of additional stress fibers in the cells. Ulinastatin treatment moderated these phenomena. The immunocytochemistry assays and western blots showed that the expression of RhoA and ROCK2 was significantly upregulated in cells treated with TNF-alpha (P < 0.05); however, ulinastatin could inhibit the high expression of these two proteins. Under treatment with TNF-alpha and ulinastatin, compared with normal EA.hy926 cells, overexpression of RhoA upregulated expression of RhoA, ROCK2 and p-MYPT1, downregulated expression of VE-cadherin, and restored the hyperpermeability of vascular endothelial cells due to TNF-alpha treatment (P < 0.05). Conclusions: Ulinastatin inhibited the hyperpermeability of vascular endothelial cells induced by TNF-alpha. This inhibitory effect of ulinastatin may be related to the RhoA/ROCK signaling pathway. (C) 2017 Elsevier B.V. All rights reserved.