Objective To investigate the protective effects of 6-O-desulfated heparin (6-DeH) on lung injury in septic mice by inhibiting the degradation of endothelial glycocalyx degradation. Methods BALB/c mice were divided into a sham group, a sepsis group, and a sepsis+6-DeH group. Cecal perforation surgery was used to establish sepsis model. After modeling, the sepsis+6-DeH group was given 2 mL of 5 g/L 6-DeH solution for nebulized inhalation, while the sham group and the sepsis group were given 2 mL of normal saline for nebulized inhalation, once every other day. Lung wet/dry weight ratio, protein concentration in bronchoalveolar lavage fluid (BALF), pathological changes of lung tissue, protein expression levels of CD31, thromboxomodulin (TM), hyaluronic acid (HA), heparan sulfate (HS), syndecan-1, glypican, angiopoietin 1 (Ang1), angiopoietin 2 (Ang2), Tie2 in lung tissue, co-localization of CD31 and syndecan-1 were detected at 14 days of intervention. Results Lung wet/dry weight ratio, protein concentration in BALF, protein expression level of Ang2 in lung tissue of the sepsis group were all significantly higher than those of the sham group, while protein expression levels of CD31, TM, HA, HS, syndecan-1, glypican, Ang1 and Tie2 in lung tissue were significantly lower than those of the sham group (all P<0.05). Lung wet/dry weight ratio, protein concentration in BALF, protein expression level of Ang2 in lung tissue of the sepsis+6-DeH group were all lower than those of the sepsis group, while protein expression levels of CD31, TM, HA, HS, syndecan-1, glypican, Ang1 and Tie2 in lung tissue were all higher than those of the sepsis group (all P<0.05). Immunofluorescence revealed co-localization of CD31 and syndecan-1 in lung tissue. Conclusions 6-DeH alleviates lung injury and inhibits degradation of endothelial glycocalyx in pulmonary blood vessels in septic mice. This effect is related to the regulation of the Ang/Tie2 pathway.