Objective To investigate the anti-inflammatory activity of Ageratina adenophora essential oil (AAEO-CP) and its effects on the expression of Toll-like receptor 4 (TLR4) protein in lipopolysaccharide (LPS)-induced RAW264.7 cells. Methods RAW264.7 cells were divided into control group, LPS group, and LPS combined with AAEO-CP group. The cytotoxicity of AAEO-CP was detected by CCK-8 assay. The mRNA and protein expression of interleukin-6 (IL-6) and IL-10 were detected by real-time PCR and ELISA, respectively, and the protein expression of TLR4 in RAW264.7 cells was measured by Western blotting. Results AAEO-CP below 20 mg/mL was not cytotoxic to RAW264.7 cells. LPS increased the protein expression of TLR4, also increased the protein and mRNA expression of IL-6, but decrease the protein and mRNA expression of IL-10 in RAW264.7 cells. And all of the above results were reversed by AAEO-CP. Conclusion AAEO-CP can play the anti-inflammatory effects by increasing the expression of IL-10 protein and decreasing the expression of IL-6 protein, and inhibiting TLR4 protein in LPS-induced RAW264.7 cells.