Objective: To investigate the protective effect of isorhamnetin on hyperoxia induced acute lung injury (ALI) in rats and its possible mechanism. Methods: Thirty-two SD rats were randomly divided into normal control group, model group, isorhamnetin group and isorhamnetin+resatorvid group [resatorvid (TAK-242) is a specific inhibitor of TLR4], with 8 rats in each group. Normal control group was fed normally, other groups of rats were placed in the self-made hyperoxia box for 3 days to construct ALI model. After the successful construction of models, rats were injected intraperitoneally according to the dosage of each group. Model group and normal control group were injected intraperitoneally with the same volume of normal saline. Each group was injected once a day, and lasted for 7 days. Abdominal aorta blood, bronchoalveolar lavage fluid and lung tissue of rats were collected, partial pressure of oxygen (PaO2) and partial pressure of carbon dioxide (PaCO2) were measured by blood gas analyzer, wet weight/dry weight (W/D) of lung tissue was measured by oven method, pathological changes of lung tissue in rats were observed by HE staining, and lung tissue injury score was performed. Expression of TLR4 of lung tissue in rats was observed by immunofluorescence, levels of inflammatory factors in bronchoalveolar lavage fluid were detected by ELISA, expressions of Toll like receptor 4/nuclear factor-κB (TLR4/NF-κB) pathway related proteins in lung tissue of rats were determined by Western blot. Results: Compared with normal control group, PaO2 value of model group was significantly decreased, PaCO2 and W/D were significantly increased, lung tissue injury was serious, the lung injury score, levels of inflammatory factors in bronchoalveolar lavage fluid and expressions of TLR4/NF-κB pathway related protein in lung tissue were significantly increased; compared with model group, lung tissue damage in isorhamnetin group and isorhamnetin+resatorvid group were alleviated, and PaO2 value was significantly increased, PaCO2, W/D value, lung injury score, levels of inflammatory factors in bronchoalveolar lavage fluid and expressions of TLR4/NF-κB pathway related proteins in lung tissue were significantly decreased; compared with isorhamnetin group, lung tissue damage in isorhamnetin+resatorvid group was alleviated, and PaO2 value was significantly increased, PaCO2, W/D value, lung injury score, levels of inflammatory factors in bronchoalveolar lavage fluid and expressions of TLR4/NF-κB pathway related proteins in lung tissue were significantly decreased. Conclusion: Isorhamnetin can alleviate hyperoxiainduced ALI in rats by inhibiting TLR4/NF-κB pathway. [ABSTRACT FROM AUTHOR]