Objective To investigate the protective effects of Matricarla chamomilla L. extract on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and explore the mechanism by which Matricarla chamomilla L. extract alleviates ALI. Methods Forty-eight male SPF Kunming mice were randomly divided into control group, LPS group, dexamethasone (DEX) group, and low, middle and high doses of Matricarla chamomilla L. extract groups. The mice in each group were first continuously administered by gastric gavage for 7 days, and dexamethasone was administered intraperitoneally in the dexamethasone group 5 mg/kg. Matricarla chamomilla L. extract was administered 85, 170 and 340 mg/(kg·d) for gastric lavage in the low, medium and high dose groups, respectively. Mice of all groups, except those of control group, were induced into ALI mouse model by intratracheal instillation of LPS. Subsequently, the mice were subjected to the determination of their levels of the inflammatory factors tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and interleukin 1β (IL-1β) in bronchoalveolar lavage fluid (BALF) and perform Giemsa staining and white blood cell count on the BALF sediment. The content and activities of malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were detected. Lung tissue was taken and the pathological morphology of the inferior lobes of the right lung was observed. The expression levels of Toll-like receptor 4 (TLR4), recombinant myeloid differentiation factor 88 (MyD88), nuclear factor-kappa B (NF-κB) in lung tissue were detected. Results Compared with the blank group, the alveolar lavage fluid of LPS model group mice TNF-α, IL-6, IL-1β, White blood cell count and serum MDA content significantly increased (P<0.05), serum SOD significantly decreased (P<0.05), HE staining showed severe interstitial inflammation infiltration in the lungs, structural damage and abnormal morphology of lung tissue, protein expression levels of TLR4, MyD88, NF-κBp65 in lung tissue of mices increased (P<0.05). Compared with the LPS model, the levels in mice of each treatment group TNF-α, white blood cell count, and serum MDA contents were significantly reduced (P<0.05), while serum SOD content was significantly increased (P<0.05). HE staining showed a decrease in inflammatory cell infiltration in mouse lung tissue, clearer alveolar spaces, more complete lung tissue morphology, and protein expression levels of TLR4, MyD88 and NF-κBp65 in lung tissue of mice decreased (P<0.05). Conclusion Matricarla chamomilla L. extract can alleviate LPS induced pulmonary inflammation and overactivation of oxidative stress, thereby alleviating ALI in mice. Among them, a dose of 170 mg/(kg·d) has a better effect, and pathway such as TLR4/MyD88/NF-κB.