<p>Mechanical ventilation (MV) is essential for patients who require life support, but undue mechanical stress leads to airway and alveolar injury, also known as ventilator-induced lung injury (VILI). MV induces changes in pulmonary endothelial barrier integrity by affecting cell junction proteins. The mechanisms of disruption of endothelial barrier integrity during VILI are still unclear. This study aimed to investigate the roles and mechanisms by which ASK1-interacting protein-1 (AIP1), G-protein pathway suppressor 2 (GPS2), and sirtuin 1 (SIRT1) affect VILI. Human lung microvascular endothelial cells (HLMVECs) were transfected with AIP1 small interfering RNA (siRNA), GPS2 siRNA, GPS2 cDNA, and SIRT1 siRNA and subjected to 20% cyclic stretch (CS). C57BL/6N mice were pretreated with the SIRT1 siRNA before MV. We found that CS of 20% activated oxidative stress, increased the reactive oxygen species (ROS) production, and disrupted the pulmonary endothelial cell barrier integrity. AIP1 depletion increased the ROS production and aggravated the disruption of endothelial barrier integrity. Loss of GPS2 decreased the level of AIP1, leading to low expression levels of cell junction proteins. These effects were alleviated by GPS2 overexpression. SIRT1 depletion induced a decrease in GPS2 and AIP1, and increased the ROS production, resulting in decreased expression levels of cell junction proteins. Furthermore, VILI was exacerbated by increased cytokine production (IL-6 and IL-1β), pulmonary oedema, and an elevated wet/dry weight ratio in SIRT1-depleted mice under MV. These results suggest that cyclic mechanical stretching activated oxidative stress and disrupted the expression of cell junction proteins. The SIRT1/GPS2/AIP1 axis influences the production of ROS to regulate the pulmonary endothelial cell barrier integrity during VILI.</p>