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Volume resuscitation is an important early treatment for haemorrhagic shock. Haemoglobin-based oxygen carrier (HBOC) can expand the volume and provide oxygen for tissues. Vascular leakage is common complication in the process of haemorrhagic shock and resuscitation. The aim of this study was to observe the effects of HBOC (a bovine-derived, cross-linked tetramer haemoglobin oxygen-carrying solution, 0.5 g/L) on vascular leakage in rats after haemorrhagic shock. A haemorrhagic shock rat model and hypoxic vascular endothelial cells (VECs) were used. The role of intercellular junctions and endothelial glycocalyx in the protective effects of HBOC and the relationship with mitochondrial function were analysed. After haemorrhagic shock, the pulmonary vascular permeability to FITC-BSA, Evans Blue was increased, endothelial glycocalyx was destroyed and the expression of intercellular junction proteins was decreased. After haemorrhagic shock, a small volume of HBOC solution (6 ml/kg) protected pulmonary vascular permeability, increased structural thickness of endothelial glycocalyx, the levels of its components and increased expression levels of the intercellular junction proteins ZO-1, VE-cadherin and occludin. Moreover, HBOC significantly increased oxygen delivery and consumption in rats, improved VEC mitochondrial function and structure. In conclusion, HBOC mitigates endothelial leakage by protecting endothelial glycocalyx and intercellular junctions through improving mitochondrial function and tissue oxygen delivery.