Abstract: Omega-3 fatty acids have natural anti-inflammatory properties, and alpha-linolenic acid (ALA) is an essential omega-3 fatty acid. In order to elucidate its anti-inflammatory mechanism in vitro, lipopolysaccharide (LPS) was used to stimulate macrophages to construct an inflammatory model. The levels of monocyte chemotactic protein (MCP-1), interleukin4 (IL-4), interleukin13 (IL-13), the production of reactive oxygen species (ROS) and the expression of related proteins in inflammatory signaling pathways were determined to explore the mechanism of α-linolenic acid in alleviating lipopolysaccharide-induced inflammatory response. The results showed that α-linolenic acid significantly inhibited the release of MCP-1 and increase the secretion level of anti-inflammatory cytokine IL-13. At the same time, α-linolenic acid also reduced ROS production and alleviated ROS-mediated inflammatory damage. In addition, α-linolenic acid could reduce the accumulation of MDA by increasing the expression of antioxidant enzymes, thereby reducing the concomitant harm of oxidative stress in inflammatory responses. Western blot analysis further confirmed that α-linolenic acid inhibited the expression of TLR4, MyD88, and downstream phosphorylation levels of p65 and IκBα. These results indicate that α-linolenic acid can significantly inhibit the LPS-induced inflammatory response of RAW264.7 cells, and the mechanism may be related to inhibiting the activation of TLR4/MyD88/NF-κB signaling pathway.