Natural polysaccharides named PEP-0.1-1, PEP-0-1 and PEP-0-2 from edible mushroom species Pleurotus eryngii were obtained in the present study. Results showed that molecular weights of these polysaccharides were 3235 kDa, 2041 kDa and 23933 Da, respectively. Further, structural characterization revealed that PEP-0.1-1 had a →4-α-D-Glcp-1→ backbone and contained →4)-α-D-Glcp and →4)-β-D-Glcp reducing end groups. PEP-0-1 backbone contained →4-α-D-Glcp-1→ and →6-α-3-O-Me-D-Galp-1→, and the side chains contained α-D-Glcp, β-DManp-1→ and α-D-Glcp-3→. However, PEP-0-2 backbone consisted of →4-α-D-Glcp-1→ and →6-α-3-O-Me-DGalp-(1→6)-α-D-Galp-1→ while the side chains contained α-D-Glcp and β-D-Manp-1→. Biological activity analysis was then carried out and found that all these polysaccharides could significantly suppress the relative mRNA expression of TLR4, nitric oxide (NO), TNF-α, IL-1β and IL-6 in lipopolysaccharide (LPS)-induced inflammation of RAW 264.7 cells, as well as the over secretion of the above cell cytokines. Moreover, western blotting analysis revealed that all these purified fractions displayed significant inhibition effects on the expression of JNK protein induced by LPS in MAPK pathway, along with the relieving on the inhibition effect of LPS on IκB-α protein expression. In summary, the information generated by the present study could provide a theoretical basis for the exploration of novel healthy food materials from edible mushrooms with anti-inflammation activities.