BACKGROUND: Mechanical ventilation (MV) is frequently used but can aggravate or cause lung injury, known as ventilator-induced lung injury (VILI). However, the mechanisms are unclear. The NLR family pyrin domain containing 3 (NLRP3) inflammasome is a vital component of innate immunity and is closely related to VILI. METHODS: Mouse lung epithelial (MLE-12) cells were transfected with NLRP3 small interfering RNA (siRNA) or scramble siRNA (sc siRNA) and subjected to 20% cyclic stretch (CS). Wild-type C57BL/6 mice were injected with a liquid complex of NLRP3 siRNA/sc siRNA-Lipofectamine 2000 through the fundus venous plexus before mechanical ventilation. Western blots, immunoprecipitation, ELISAs, flow cytometry, immunofluorescence, and hematoxylin-eosin staining were used to assess the effects of the NLRP3 inflammasome on VILI and the mechanisms of those effects. RESULTS: CS activated the NLRP3 inflammasome by activating NIMA-related kinase 7 (NEK7). NLRP3 depletion inhibited NLRP3 inflammasome activation; alleviated the degradation of cell junction proteins, including p120-catenin (p120) and occludin; ameliorated the colocalization of p120 and E-cadherin; and mitigated the decrease in mitochondrial membrane potential caused by mechanical stretch. Furthermore, after NLRP3 depletion, VILI was attenuated by decreasing IL-1β secretion and pulmonary edema. CONCLUSIONS: Inhibiting NLRP3 inflammasome activation ameliorated VILI, suggesting a potential therapeutic target for the clinical treatment of VILI.