Abstract: OBJECTIVE To explore the correlation between Toll-like receptor 4/nuclear factor-κB（TLR4/NF-κB） signaling pathway and levels of inflammatory cytokines in children with obesity and Mycoplasma pneumonia（MP） infection. METHODS A total of 68 children with obesity and MP infection admitted to Huaian Second People’s Hospital were enrolled as the study group between May 2018 and Aug 2020; 60 obesity children admitted to the hospital and 60 healthy children undergoing physical examination during the same period were enrolled as the obesity group and healthy group, respectively. The relative mRNA expression levels of TLR4, NF-κB and NF-κB inhibitory protein（IκB） in peripheral blood, and levels of serum interleukin-6（IL-6）, interleukin-8（IL-8） and tumor necrosis factor-α（TNF-α） were detected and compared among the three groups. The relationship between levels of TLR4, NF-κB and IκB and serum inflammatory cytokines such as IL-6, IL-8 and TNF-α was analyzed. The risk factors for MP infection in obesity children were analyzed by univariate and multivariate Logistic regression analysis. RESULTS There were significant differences in the relative mRNA expression levels of TLR4, NF-κB and IκB, and levels of serum IL-6, IL-8 and TNF-α among the three groups（P＜0.05）. The relative mRNA expression levels of TLR4 and NF-κB, and levels of serum IL-6, IL-8 and TNF-α in the study group and obesity group were significantly higher than those in the healthy group, while IκB mRNA was significantly lower than that in the health group（P＜0.05）. The relative mRNA expression levels of TLR4 and NF-κB in the study group were significantly higher than those in the obesity group, while IκB mRNA was significantly lower than that in the obesity group（P＜0.05）. The correlation analysis showed that the expressions of TLR4 and NF-κB were significantly positively correlated with IL-6, IL-8 and TNF-α（P＜0.05）, while IκB was significantly negatively correlated with them（P＜0.05）. Multivariate Logistic regression analysis showed that age within 3-7 years old, low complement status and history of epidemic exposure were independent risk factors for MP infection in the obesity children. CONCLUSION TLR4/NF-κB signaling pathway in obesity children combined with MP infection is over-activated, which may promote the release of IL-6, IL-8, TNF-α and other inflammatory cytokines, and thus inducing pulmonary inflammation response.