Acute lung injury (ALI), hallmarked with alveolar epithelial barrier impairment and pulmonary edema induced by acute inflammation, presents a severe health burden to the public, due to the limited available interventions. Oxyberberine (OBB), having improved anti-inflammatory activity and safety, is a representative component with various activities derived from berberine, whereas its role against ALI with alveolar epithelial barrier injury remains uncertain. To investigate the influence and underlying mechanisms of OBB on ALI, we induced acute inflammation in mice and A549 cells by using lipopolysaccharide (LPS). Changes in alveolar permeability were assessed by analyzing lung histopathology, measuring the dry/wet weight ratio of the lungs, and altering proinflammatory cytokines and neutrophils levels in the bronchoalveolar lavage fluid (BALF). Parameters of pulmonary permeability were assessed through ELISA, western blotting, quantitative real-time PCR, and immunofluorescence analysis. U46619, the agonist of RhoA/ROCK, was employed to further investigate the mechanism of OBB on ALI. Unexpectedly, we found OBB mitigated lung impairment, pulmonary edema, inflammatory reactions in BALF and lung tissue, reduction in ZO-1, and addition of connexin-43. Besides, OBB markedly reduced the expression of RhoA in association with its downstream factors, which are linked to the intercellular junctions and permeability both in vivo and in vitro. Nevertheless, U46619 abolished the benefits obtained from OBB in A549 cells. In conclusion, these outcomes indicated that OBB exerted RhoA/ROCK inhibitor-like effect to moderate alveolar epithelial barrier impairment and permeability, ultimately preventing ALI progression. Display omitted •OBB protected ALI through maintenance of the alveolar epithelial barrier.•OBB attenuated pulmonary edema via modulating barrier proteins connexin-43 and ZO-1.•OBB mediated pulmonary protection by inhibiting the RhoA/ROCK signaling pathway.