Pleurotus eryngii polysaccharide (PEP) was obtained using hot water extraction and ethanol precipitation. Three purified polysaccharide fractions (namely PEP1-A, PEP2-A and PEP3-A) were obtained from PEP using DEAE-cellulose-52 chromatography and a gel permeation Sephadex G-100 column. Firstly, this paper examined the characterisation of PEP1-A, PEP2-A and PEP3-A. The corresponding molecular weights were 5.378 9 10 5 , 9.506 9 10 6 and 4.975 9 10 5 Da, respectively. PEP1-A, PEP2-A and PEP3-A had similar monosaccharide compositions. PEP1-A was b-configuration, and PEP2-A and PEP3-A were a-configuration. PEP1-A, PEP2-A and PEP3-A had pyran-type rings, (1 ? 3) glucose and (1 ? 6) galactose linkages. Secondly, PEP, PEP1-A, PEP2-A and PEP3-A possessed antioxidant activities, and PEP was best. Therefore, only PEP was used to study its anti-fatigue activity in vivo. The result proved that PEP had anti-fatigue activity. PEP could be used as a valuable natural food supplement for preventing anti-fatigue or functional food.