A novel water-soluble polysaccharide from Pleurotus eryngii residue was isolated and further purified by DEAE cellulose-52 chromatography and Sephadex G-100 size-exclusion chromatography to yield PEPE-1, PEPE-2 and PEPE-3. Molecular weights were determined by high-performance size-exclusion chromatography (HPSEC). Gas chromatography (GC) analysis of monosaccharide composition confirmed that PEPE-1, PEPE-2 and PEPE-3 were heteropolysaccharides and mainly composed of glucose. Sulfate and uronic acid content, ultraviolet and infrared spectrum were also evaluated. The antitumor activities of the polysaccharides against HepG-2 cells were studied in vitro. Results showed that the three polysaccharides could suppress the proliferation and enhance lactate dehydrogenase (LDH) release of HepG-2 cells in a dose- and time-dependent manner. The effect increased in the order of PEPE-1 < PEPE-2 < PEPE-3, respectively. The same order was also observed with uronic acid content. Findings presented in this study suggested that the polysaccharides extracted from P. eryngii residue might be suitable for functional foods and natural antitumor drugs development.