Introduction: Over decades, the source-function relationships of bioactive polysaccharides have been progressively investigated, however, it is still unclear how a defined structure may conduce to the bioactivities of polysaccharides. Objectives: To explore the structure–function relationship of fungus polysaccharides, we employed a dextran sulfate sodium (DSS)‐induced colitis mouse model to compare the anti-inflammatory activity of two fungus polysaccharides from Dictyophora indusiata (DIP) and Tremella fuciformis (TFP), which exhibit distinct glycosidic linkages. Methods: The structures of DIP and TFP were characterized through molecular weight detection, molecular morphology analysis, methylation analysis, and NMR analysis. Subsequently, we employed a DSS-induced colitis model to assess the anti-inflammatory efficacy of DIP and TFP. The colitis symptoms, histological morphology, intestinal inflammatory cytokines, and the composition and function of gut microbiota before and after polysaccharides treatment in colitis mice were also investigated. Results: DIP, l,3-β-D-glucan with 1,4-β and 1,6-β-D-Glcp as branched chains, exhibited superior therapeutic effect than that of TFP consisted of a linear 1,3-α-D-mannose backbone with D-xylose and L-fucose in the side chains. Both DIP and TFP relieved DSS-induced colitis in a gut microbiota-dependent manner. Furthermore, metagenomics showed that DIP and TFP could partially reverse the bacterial function in colitis mice. Glycoside Hydrolase 1 (GH1) and GH3 were identified as being involved in hydrolyzing the glucose linkages in DIP, while GH92 and GH29 were predicted to be active in cleaving the α-1,3-linked mannose linkages and the glycosidic bonds of L-fucose residues in TFP. Conclusion: Our findings highlight the pivotal role of glycosidic linkages in anti-inflammatory activities of fungus polysaccharides and would promote the design and discovery of polysaccharides with designated activity to be used as functional foods and/or therapeutics.