The aim of this study was to evaluate the spatial structure and potential antifatigue activity of polysaccharide fractions which was extracted from Inonotus obliquus. The first polysaccharide fractions of Inonotus obliquus (PIO-1) were obtained after hot-water extraction and purification by DEAE cellulose-52 chromatography. Results of the forced swimming test showed that the doses (50 mg/kg) of PIO-1 could increase the climbing duration and swimming time as well as reduced the immobility time in the PIO treated mice. The fatigue related metabolic parameters showed that PIO-1 decreased the level of blood lactic acid (BLA), urea nitrogen (BUN) and lactic dehydrogenase (LDH). Additionally, PIO-1 significantly decreased the 5-HT concentrations in the mice brain. The results of monosaccharide analysis showed that the molar ratio of mannose, glucose, galactose, xylose and arabinose with the molar ratio of 1.0:1.9:3.5:18.5:5.7. The molecular morphology of the PIO-1 observed under atomic force microscopy (AFM). There were many spherical and heterogeneous clumps existed in the images. Therefore. current study indicated polysaccharide PIO-1 not only has great potential to postpone physical fatigue but also shown potential to improve mental fatigue. (C) 2020 Elsevier B.V. All rights reserved.