Abstract: A neutral polysaccharide was purified by DEAE-52 cellulose column chromatography from the crude polysaccharide of Arctium lappa L. obtained by hot water extraction and ethanol precipitation, and the structure and anti-inflammatory activity of the neutral polysaccharide were characterized. The structure of Arctium lappa L. neutral polysaccharide were investigated through monosaccharide composition, molecular weight, ultraviolet spectra, infrared spectroscopy analysis, X-ray diffraction spectroscopy, particle size analysis and NMR analysis. The effect of different doses of crude polysaccharide and neutral polysaccharide from Arctium lappa L. on macrophage movement was evaluated in zebrafish acute inflammation model induced by copper sulfate. The mRNA expressions of different inflammatory factors such as nuclear transcription factor activator protein-1 (AP-1), cycloxygenase-2 (COX-2), IKBα, interleukin-1β (IL-1β), interleukin-12 (IL-12) and myeloid differentiation factor (MyD88) were determined by real-time PCR analysis. Results showed that the weight-average molecular weight and number-average molecular weight of Arctium lappa L. neutral polysaccharide were 2682 and 2079 Da, which were mainly composed of fructose and glucose with a molar ratio of 11.32:1. The particle size mainly ranged from 80 nm to 110 nm. The neutral polysaccharide of Arctium lappa L. was mainly composed of furanose with β configuration, with obvious sharp and narrow crystal diffraction peaks and high crystallinity. The NMR analysis showed that its structure was α-D-glucopyranosyl-(1→2)-β-D-fructofuranosyl-(1→2)₁₀-β-D-fructofuranosyl. Arctium lappa L. neutral polysaccharide could significantly (P<0.05) reduce the migration number of zebrafish macrophages, and the anti-inflammatory effect was the best at the concentration of 100 µg/mL, as well as the number of macrophages decreased by 41.11% compared with the model group. The Arctium lappa L. neutral polysaccharide may be inhibited the overactivation of TLR4/MyD88/NF-κB signaling pathway by silting the signal transduction of inflammatory cytokines. This study could provide a theoretical basis for the anti-inflammatory activity of neutral polysaccharide from Arctium lappa L. and the development of functional foods.