The anti-inflammatory effects and mechanisms of the mycelial polysaccharide ISP-80 from the Isaria cicada Miquel C1 was investigated in a mouse monocyte-macrophage RAW264.7 inflammation model, induced by lipopolysaccharide (LPS). The polysaccharide (ISP-80) was obtained through hot water extraction from the Isaria cidada C1 mycelium. The toxic effects of ISP-80 on RAW264.7 cells were evaluated using the CCK-8 assay, and the impact of ISP-80 on the secretion of TNF-α, IL-1β, and IL-6 cytokines was measured using ELISA. Furthermore, the mechanism of ISP-80 was analyzed using western blotting to detect the relevant proteins in the MAPK/NF-κB pathways. The findings indicated that the extraction rate of ISP-80 was 10.42% and it was composed of three polysaccharide fractions, each containing Glc, Man, and Gal monosaccharides, in a molar ratio of 1.00:5.03:1.55. Adding ISP-80 at 0.01~1.00 mg/mL exerted no cytotoxic effects and promoted cell proliferation (P<0.000 1). The 0.25 mg/mL ISP-80 treatment significantly decreased the secretion of pro-inflammatory cytokines IL-1β (P<0.001) and IL-6 (P<0.05), reaching 0.59- and 0.88-times those of the LPS group, respectively, and inhibited cell differentiation while restoring cell morphology. The anti-inflammatory action of ISP-80 acted via the MAPK/NF-κB pathways. ISP-80 inhibited the MAPK pathway by blocking p-p38 and p-JNK, which decreased by 0.20 and 0.41, respectively (P<0.001). Furthermore, the NF-κB pathway was inhibited by blocking IκBα and p65, which decreased by 0.43 (P<0.01) and 0.24 (P<0.001), respectively. These results indicate that ISP-80 can be a natural and safe antiinflammatory agent for the prevention and treatment of inflammatory diseases.