Abstract: Objective To explore the effects of Thymosin β4 （Tβ4） on the lipopolysaccharide （LPS）-induced polarization tendency of murine monocyte-macrophage RAW264.7 cells and its influence on inflammatory responses. Methods An inflammation model was established by LPS induction in RAW264.7 cells. Cell viability was assessed using the CCK-8 assay. The concentrations of cytokines tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） were detected by enzyme-linked immunosorbent assay （ELISA）. Nitric oxide （NO） secretion in the cell culture supernatant was detected using the Griess method. Real-time quantitative polymerase chain reaction （RT-qPCR） was employed to detect mRNA expression levels of nuclear factor-κB （NF-κB）, cyclooxygenase-2 （COX-2）, TNF-α, and IL-6. Western blotting was used to analyze protein levels of inducible nitric oxide synthase （iNOS）, NF-κB, phosphorylated NF-κB （p-NF-κB）, NF-κB inhibitor α （IκB-α）, and phosphorylated IκBα （p-IκB-α）. The polarization status of macrophages was observed using double fluorescence staining. Immunofluorescence staining was performed to examine NF-κB localization and expression. Results After treatment with 1 000 μg/L Tβ4 for 24 hours, the cell viability of RAW 264.7 cells was 90.2%, showing a statistically significant difference compared to the blank control group （P < 0.05）. Tβ4 at various concentrations effectively inhibited NO production （all P < 0.000 1）. Tβ4 decreased the concentrations of pro-inflammatory cytokines（TNF-α and IL-6） and NO in the LPS-induced RAW264.7 inflammatory model. It also downregulated mRNA expression of NF-κB, COX-2, TNF-α, and IL-6, as well as protein expression of iNOS, p-NF-κB, and p-IκBα. Additionally, Tβ4 inhibited NF-κB nuclear translocation and decreased CD80 expression （all P < 0.05）. Conclusion Tβ4 exhibits anti-inflammatory effects, the mechanisms of which may be associated with the inhibition of the NF-κB signaling pathway and suppression of macrophage M1 polarization.