Objective To explore the regulatory effect of quorum sensing molecule N-3-oxodecanoyl-L-homoserine lactone (3-oxo-C10-HSL) on lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophages. Methods RAW264.7 macrophages were divided into experimental group, control group and blank group. The experimental group was treated with different concentrations of 3-oxo-C10-HSL and LPS; the control group was treated with DMSO and LPS; and the blank group was treated with DMSO and PBS. Cells and supernatants were collected after 12 hours of stimulation. The mRNA expression of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) were detected by real-time quantitative PCR, and the protein levels of IL-6 and TNF-α in supernatant were detected by ELSIA. Further, 25 μmol/L 3-oxo-C10-HSL and 100 ng/mL LPS were used to stimulate the cells for 15, 30 and 60 minutes, and the phosphorylation of nuclear factor κBp65 (NF-κBp65) was detected by Western blot analysis. Results The 3-oxo-C10-HSL could decrease the mRNA levels of IL-6, IL-1β, TNF-α, MCP-1 and the protein levels of IL-6 and TNF-α in LPS-treated RAW264.7 macrophages in a dose-dependent manner. In addition, 3-oxo-C10-HSL could inhibit the phosphorylation of NF-κBp65 induced by LPS. Conclusion 3-oxo-C10-HSL can alleviate LPS-induced inflammatory responses in RAW264.7 macrophages by inhibiting activation of NF-κB signaling pathway.