Purpose <p>This study aimed to investigate the involvement of angiopoietin (Ang)/Tie2 pathway in mediating pulmonary endothelial glycocalyx injury in histone-induced acute lung injury in mice, and the protective mechanism of unfractionated heparin (UFH).</p> Methods <p>Twenty-four male C57BL/6 mice (20–25&#xa0;g), 8–12 weeks old, were randomly divided into control, histone, and histone + UFH groups. The histone (50&#xa0;mg/kg) was administered via tail vein. UFH (400 U/kg) was administered 1&#xa0;h after histone injection. The control group was administered by an equal amount of sterile saline solution. The lungs of all groups were harvested 4&#xa0;h after the injection of histones or sterile saline.</p> Results <p>UFH attenuated histone-induced lung histopathological changes and edema. UFH alleviated pulmonary endothelial injury and glycocalyx shedding by reducing histone-induced low expression of thrombomodulin (TM) and decreased lung syndecan-1 levels. UFH improved histone-induced low mRNA expression of TM, syndecan-1, Ang-1, Tie2 and high expression of heparinase (HPA), Ang-2.</p> Conclusion <p>UFH may attenuate histone-induced lung injury and pulmonary endothelial glycocalyx degradation via the Ang/Tie2 pathway.</p>