This study investigated the effects of heat treatment on structural characteristics and in vitro antitumor activity of polysaccharides from Grifola frondosa. GFP-4 (extracted at 4 degrees C), GFP-4-80 (80 degrees C treatment on GFP-4) and GFP-80 (extracted at 80 degrees C) were prepared, and the chemical composition analysis showed that their total sugar contents were all higher than 90%, high-performance gel-permeation chromatography (HPGPC), ion chromatography (IC) and Fourier-transform infrared spectroscopy (FTIR) results demonstrated that GFP-4 were degraded and denatured after 80 degrees C heat treatment, MTT and JC-1 results showed that GFP-4 exhibited higher inhibitory effects on HepG2 cells in vitro than GFP-4-80 and GFP-80. Our study suggested that heat treatment at 80 degrees C on polysaccharides from Grifola frondosa would destroy their structure and attenuate their antitumor effects.