Objective To investigate the effect of ROCK inhibitor fasudil (FS) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and its possible mechanisms. Methods Forty male C57BL/6 mice were randomly divided into four groups (n=10): control group, ALI group, FS group, and ALI+FS group. After administration of LPS or FS, lung histopathological changes, inflammatory cytokine levels, expression levels of Rho kinase (ROCK), and cell pyroptosis-related proteins, including NLRP3, ASC, caspase-1, cleaved caspase-1, GSDMD, cleaved GSDMD, and IL-1β, were assessed. In vitro, human umbilical vein endothelial cells (HUVECs) were cultured and the cell activity, LDH release rate, inflammatory factor levels, and the expression levels of cell pyroptosis-related proteins were measured after administration of LPS+ATP and FS. Results In vivo experiments on mice demonstrated that the alveolar hemorrhage, alveolar septum thickening, and lung tissue edema were more pronounced in ALI group compared to the control group, and FS pretreatment significantly reduced these histopathological changes. Compared with the control group, mice in ALI group showed increased lung wet/dry weight ratio, increased total cell count and total protein concentration in alveolar lavage fluid, increased levels of TNF-α, IL-6, IL-18, and IL-1β, and increased expression of ROCK1, ROCK2, and pyroptosis-related proteins including NLRP3, ASC, cleaved caspase 1, cleaved GSDMD, and IL-1β in pulmonary tissues (P<0.05); compared with mice in ALI group, mice in ALI+FS group had a lower lung wet/dry weight ratio, a lower total cell count and total protein concentration in alveolar lavage fluid, lower levels of inflammatory factors and lower expression levsls of ROCK1, ROCK2, and pyroptosis-related proteins in lung tissue (P<0.05). In vitro experiments on HUVECs cells confirmed that compared with the control group, ALI group showed a significant decrease in cell viability, an increase in LDH release rate, cell death rate, and inflammatory factor levels, and an upregulation of the expression of the pyroptosis-related proteins (P<0.05); compared with ALI group, ALI+FS group showed an increase in cell viability, a significant decrease in LDH release rate, cell death rate, and inflammatory factor levels, and downregulation of the expression of pyroptosis proteins (P<0.05). Conclusion FS may alleviate LPS-induced ALI by inhibiting endothelial cell pyroptosis.