Abstract: To systematically investigate the structural features and anti-inflammatory activity of polysaccharide (USP-N) from Orchis chusua D. Don, a medicinal and edible plant was selected. The structure of USP-N was comprehensively elucidated through partial acid hydrolysis, Smith degradation, Methylation analysis, and 2D-NMR spectroscopy. Its anti-inflammatory effects were evaluated using a zebrafish inflammation model. Structural analysis revealed that the main chain of USP-N was composed of alternating β-1,4-linked glucose (38.5%) and mannose (33.8%), forming a glucomannan backbone. The side chains contain small amounts of rhamnose, arabinose, and galactose, with some mannose residues featuring 2-O-acetyl groups. Bioactivity assays demonstrated that USP-N treatment significantly suppressed LPS-induced nitric oxide (NO) release (P<0.0001) and inhibited neutrophil migration to the tail injury site in zebrafish (P<0.0001). Furthermore, dose-dependent reductions in neutrophil and macrophage recruitment to the caudal region following mechanical injury were observed. This study provides a theoretical foundation for applying Orchis chusua D. Don polysaccharides in functional foods and pharmaceutical development.