BACKGROUND Increased endothelial permeability is involved in ventilator-induced lung injury (VILI). Stim1/Orai1 mediates store-operated Ca2+ activation, which modulates endothelial permeability. However, the underlying mechanisms of the Stim1/Orai1 pathway in VILI are poorly understood. MATERIAL AND METHODS Wistar rats were exposed to low tidal volume (7 mL/kg) or high tidal volume (40mL/kg) ventilation. Human Lung Microvascular Endothelial Cells (HULEC) were subjected to 8% or 18% cyclic stretching (CS). BTP2 pretreatment was performed. Lung wet/dry weight ratio, histological changes of lung injury, and bronchoalveolar lavage fluid (BALF) protein were measured. Endothelial permeability and intracellular calcium concentration were evaluated in HULECs. Protein expression was determined by Western blotting. RESULTS High tidal volume mechanical ventilation-induced lung injury (such as severe congestion and hemorrhage) and BTP2 pretreatment protected lungs from injury. The expression of Stim1, Orai1, and PKCα, lung wet/dry weight ratio, and BALF protein level significantly increased in the high tidal volume group compared to the control group and low tidal volume group. Importantly, BTP2 pretreatment alleviated the above-mentioned effects. Compared with exposure to 8% CS, the protein levels of Stim1, Orai1, and PKCα in HULECs significantly increased after exposure to 18% CS for 4 h, whereas BTP2 pretreatment significantly inhibited the increase (P<0.05). BTP2 pretreatment also suppressed increase of endothelial permeability and the intracellular calcium induced by 18% CS (P<0.05). CONCLUSIONS When exposed to high tidal volume or large-magnitude CS, Stim1 and Orai1 expression are upregulated, which further activates calcium-sensitive PKCα and results in calcium overload, endothelial hyperpermeability, and, finally, lung injury.