Objective To observe the protective effects of unfractionated heparin (UFH) on high-mobility group box-1 protein (HMGB1) induced increased permeability of endothelial cells, and investigate the protective mechanism of UFH on HMGB1 induced defective expression of zonula occludens-1 (ZO-1). Methods Human umbilical vascular endothelial cells (HUVECs) were culturedin vitro and divided into 4 groups (n=5), namely a control group, a HMGB1 group (100 ng/ml), a heparin group (UFH 10 U/ml), a HMGB1/heparin group (100 ng/ml HMGB1 + UFH 10 U/ml). Endothelial cell viability was measured by methyl thiazolyl tetrazolium (MTT) colorimetric method. Endothelial permeability was determination by Transwell chamber method. Immunofluorescence and laser confocal microscopy were used to assess the distribution of ZO-1. The protein expressions of tight junction protein ZO-1 and nuclear factor kappa B (NF-κB) were detected by Western blot. Results HMGB1 (100 ng/ml) had no inhibitory effect on endothelial cell viability (P＞0.05). UFH pretreatment could reduce the permeability increment of endothelial cells induced by HMGB1. UFH pretreatment could reduce the close loop reduction and damage of ZO-1 induced by HMGB1, enhance the fluorescence intensity and expression of ZO-1, and decrease the NF-κB translocation. Conclusions UFH can protect HMGB1-mediated defect of ZO-1 expression and increased permeability of the endothelial cells. The mechanism may be related to the decreased nuclear translocation of NF-κB.