Multiple organ perfusion is impaired in sepsis. Clinical studies suggest that persistent perfusion disturbances are prognostic of fatal outcome in sepsis. Pyroptosis occurs upon activation of caspases and their subsequent cleavage of gasdermin D (Gsdmd), resulting in Gsdmd-N (activated NH-terminal fragment of Gsdmd) that form membrane pores to induce cell death in sepsis. In addition, Gsdmd mice are protected from a lethal dose of lipopolysaccharide (LPS). However, how Gsdmd-mediated pyroptosis occurs in endothelial cells and leads to impaired perfusion remain unexplored in endotoxemia. We used transgenic mice with ablation of Gsdmd and determined that mice lacking Gsdmd exhibited reduced breakdown of endothelial barrier, improved organ perfusion, as well as increased survival in endotoxemia. Phospholipase Cγ1 (PLCγ1) contributed to Gsdmd-mediated endothelial pyroptosis in a calcium-dependent fashion, without affecting Gsdmd-N production. Cytosolic calcium signaling promoted Gsdmd-N translocation to the plasma membrane, enhancing endothelial pyroptosis induced by LPS. We used adeno-associated virus (AAV9) vectors carrying a short hairpin RNA (shRNA) against murine PLCγ1 mRNA under control of the tie1 core promoter (AAV-tie1-sh-PLCγ1) to uniquely downregulate PLCγ1 expression in the endothelial cells. Here, we showed that unique inhibition of endothelial PLCγ1 attenuated breakdown of endothelial barrier, reduced vascular leakage, and improved perfusion disturbances. Moreover, unique downregulate endothelial PLCγ1 expression markedly decreased mortality of mice in endotoxemia. Thus, we establish that endothelial injury as an important trigger of fatal outcome in endotoxemia. Additionally, these findings suggest that interfering with Gsdmd and PLCγ1-calcium pathway may represent a new treatment strategy for critically ill patients sustaining endotoxemia. Our study newly reveals that Phospholipase Cγ1 (PLCγ1) contributes to gasdermin D (Gsdmd)-mediated endothelial pyroptosis in a calcium-dependent fashion. Cytosolic calcium signaling promotes activated NH-terminal fragment of Gsdmd (Gsdmd-N) to translocate to the plasma membrane, enhancing endothelial pyroptosis induced by cytoplasmic LPS. Genetic or pharmacologic inhibition of endothelial PLCγ1 attenuated breakdown of endothelial barrier, reduced vascular leakage, improve perfusion disturbances, and decrease mortality of mice in endotoxemia.