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In recent years, the structure of selenium-enriched polysaccharides and their application in immunomodulation have attracted much attention. In previous studies, we extracted and purified a novel selenium-enriched Pleurotus ostreatus polysaccharide called Se-POP-21, but its structure and immunomodulatory activity were still unclear. In this study, the main structural unit formula of Se-POP-21 was characterized by methylation analysis and an NMR experiment. The results showed that the backbone of Se-POP-21 was →[2,6)-α-D-Galp-(1→6)-α-D-