Asthma is a chronic respiratory disease characterized by airway inflammation. Lignosus rhinocerotis (LR), a medicinal mushroom rich in polysaccharide, has been traditionally used to treat various diseases, including asthma. This study aimed to fractionate, characterize and evaluate the anti-asthmatic effects of polysaccharides from LR (LRP). LRP was isolated and characterized using high-performance liquid chromatography (HPLC), x-ray diffraction analysis (XRD), fourier transform infrared spectrometry (FTIR) and nuclear magnetic resonance (NMR) spectroscopy. In an OVA-induced asthma model, BALB/c mice were sensitized, challenged, and intranasally treated with LRP. Experimental findings demonstrated that LRP exhibits a semi-crystalline nature with predicted structure of → 4)-α-D-Glcp-(1 → and → 3)-β-D-Glcp-(1→. LRP significantly reduced eosinophilia, Th2 cytokines, and IgE levels. Histological analysis revealed LRP's ability to decrease epithelial damage and epithelial and smooth muscle thickness in lung. Reductions in inflammatory cell infiltration, mucus production, and transforming growth factor (TGF)-β1 expression were observed, although not statistically significant. Gene expression analysis indicated that LRP significantly downregulated the inducible nitric oxide synthase (iNOS) expression. This study highlights a detailed structural analysis of LRP and its potential as an alternative for the management of asthma. Further research is needed to elucidate the precise mechanisms of action and optimize its therapeutic application.