Heat stroke is a life-threatening disease with high mortality rate and unexpected complications. Vascular endothelial glycocalyx is essential for maintaining endothelial cell structure and function as well as preventing adhesion of inflammatory cells. Potential relationship that underlays the imbalance in inflammation and coagulation remains elusive. Moreover, the role of endothelial glycocalyx in heat stroke-induced organ injury remained unclear. Heat stress and lipopolysaccharide (LPS) are employed to construct in vitro models to study the change of glycocalyx structure and function in human pulmonary microvascular endothelial cells (HPMEC), as well as levels of heparansulfate proteoglycan (HSPG), syndecans-1, heparansulfate, TNF-α, IL-6, vWF, ET-1, Occludin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and reactive oxygen species (ROS). Here, we showed that heat stress and LPS devastated endothelial glycocalyx structure, activated endothelial glycocalyx degradation, and triggered oxidative damage in addition to apoptosis in HPMEC. Stimulation of heat stress and LPS increased HSPG, syndecans-1 (SDC-1), and heparansulfate levels, and promoted the ability to produce and release pro-inflammation cytokines (TNF-α, IL-6,) and coagulative factor (vWF, ET-1) in HPMEC. Furthermore, E-selectin, VCAM-1, and ROS expression were upregulated in contrast to Occludin downregulation. These changes could be deteriorated by Heparanase, whereas could be ameliorated by unfractionated heparin. This study highlights that heat stroke-induced endothelial glycocalyx degradation can trigger oxidative and apoptosis in HPMEC, in addition to dysfunction of inhibition of inflammatory response and protection in vascular permeability.