Abstract: OBJECTIVE To explore the effects of baicalin on the Toll-like receptors 4/nuclear factor κB(TLR4/NF-κB) signaling pathway and inflammatory response in mice with Mycoplasma pneumoniae. METHODS A total of 60 BALB/c mice were divided into the control group, model group, azithromycin group, baicalin high dose group, baicalin medium dose group and baicalin low dose group by using random numerical table method. Mouse model of Mycoplasma pneumoniae infection was established by intranasal method, and baicalin and azithromycin were used for the intervention. Basic signs, lung function, pulmonary histopathological changes and inflammatory factor levels, TLR4 and NF-κB-related mRNA and protein expression levels in lung tissues of the mice were detected. RESULTS Compared with the control group, the body temperature and lung index were higher than those in the model group, the azithromycin group, the baicalin high-dose group, the baicalin medium-dose group and the baicalin low-dose group, while the body mass, and lung dry-to-wet ratio were lower (P＜0.05). Compared with the model group, the peak exhalation flow(PEF), maximal voluntary ventilation(MVV), and dynamic lung compliance(Cydn) in the azithromycin group and the baicalin high-dose, medium-dose, and low-dose groups were upregulated, while inspiratory resistance(RI), exhalation resistance(RE) and lung resistance(RL) were downregulated. Lung function was protected, and the levels of inflammatory factors in alveolar lavage fluid, serum, and lung tissue were downregulated. The degree of pathological damage to lung tissue in the azithromycin group and baicalin high-dose, medium-dose, and low-dose groups decreased. When compared with the model group, the mRNA and protein relative expression levels of TLR4, NF-κB, and human nuclear factor κB inhibitory protein α (I-κB) were downregulated in the azithromycin group and baicalin high-dose, medium-dose, and low-dose groups (P＜0.05). CONCLUSION Baicalin may inhibit inflammatory response by regulating the expression of TLR4/NF-κB signaling pathway, thereby protecting lung function and having an anti-inflammatory effect.