In this study, the structural characterization, physicochemical property, antioxidant and antitumor activity of an acidic polysaccharide (APS) from Polygonum multiflorum were investigated. Monosaccharide composition analysis showed APS was composed of arabinose, rhamnose, galactose and galacturonic acid in the molar ratio of 1.23:1.32:1.48:1.00. The presence of uronic acid was also confirmed by the bands at 1740, 1645 and 1425cm-1 on Fourier transform-infrared spectroscopy. Methylation and nuclear magnetic resonance analyses showed APS was mainly composed by the residues of →5)-α-l-Araf-(1→, →3)-β-d-Galp-(1→, →3,6)-β-d-Galp-(1→, →4)-α-d-GalAp-(1→ and →2)-α-l-Rhap-(1→ in the backbone. The non-reducing terminal α-l-Araf-(1→ was probably attached to the O-6 position of →3,6)-β-d-Galp-(1→ residues. Besides, APS exhibited rod-like and flaky shapes with rough surface. The initial decomposition of APS occurred at 172°C, and the rapidest weight loss rate of APS appeared at 320°C. Antioxidant activity assay showed the DPPH radical scavenging activity of APS was 67.5% at 1mg/mL. At the concentration of 400μg/mL, the antiproliferation activities of APS against HepG-2 and BGC-823 cells were 65.28% and 51.57%, respectively. Our results suggested APS could be a potential antioxidant and antitumor agent.