Background: Previous studies have revealed that edible fungi are rich in polysaccharides, which typically have strong biological activity. Pholiota nameko is one of the main cultivated edible fungi and an important economic crop. Objectives: In order to screen a polysaccharide with antioxidant activity from Pholiota nameko. Methods and Materials: A water-soluble acidic P. nameko polysaccharide (PNP) was found from extraction with alkaline water. After purification by chromatography, the structural features of PNP were analyzed by chromatographic, spectral, & nuclear magnetic resonance (NMR) spectroscopic analyses and the content of sulfate & uronic acid in PNP was determined concurrently. Results: The content of sulfate and uronic acid was 1.756 and 7.173 w/w, respectively. The molecular weight of PNP was 1.05 × 106 Da by gel permeation chromatography. The Fourier transform infrared (FTIR) and NMR spectra displayed that the following sugar chains might be present in PNP: β-1,4-D-Manp, →4)-β-D-Manp-(1→, β-1,3-D-Glcp, β-D-Xylp-(1→. Evaluation of in vitro antioxidant activity showed that PNP had a strong scavenging ability to scavenge ABTS+, hydroxyl, and superoxide anion radicals. The results of our in vivo experiments exposed that PNP might significantly lessen the formation of malondialdehyde. High levels of PNP can meaningfully increase the activity of catalase and superoxide dismutase activity in mice serum. In addition, PNP can significantly increase the activity of GSH-Px, effectively protect the structure and function of the cell membrane. Conclusion: PNP has antioxidant effect and can be explored as a potential antioxidant.