There has been controversy regarding the optimal processing technology that destroys the special spore wall of Ganoderma lucidum so that the functional activity of the internal polysaccharides can be maintained in the most primitive state. Here, high-purity spore polysaccharides derived from the same variety were obtained respectively by three maturation methods, including enzymatic (EW-IB), laser (LW-IB) and mechanical (MW-IB).Structural characterization showed that EW-IB mildly increased the branching degree (0.523) while preserving →3)-β-D-Glcp-(1→; LW-IB slightly reduced it (0.464) without disrupting the main chain glycosidic bonds; MW-IB partially cleaved →6)-β-D-Glcp-(1→ and moderately decreased the branching degree (0.21). Functionally, the experimental results of the LPS-induced inflammation model revealed that the obtained three polysaccharides exerted their regulatory effects by applying differential forces to specific binding sites in the cell. Remarkably, preservation of →3)-β-D-Glcp-(1→ and moderate branching degree correlated with immunomodulatory activity, while excessive glycosidic bond cleavage (MW-IB) or increased branching degree (EW-IB) slightly reduced regulation of inflammatory factors NO and IL-1β. Overall, our findings point towards a valuable approach for the high-value processing and utilization of medicinal fungal polysaccharides.