Mushroom polysaccharides isolated from fruiting bodies are known for their antitumor activities, whereas those from mushroom sclerotia are not. A hot water- (PR-HW) and a cold alkaline (PR-CA)-soluble polysaccharide isolated from a novel mushroom sclerotium, Polyporus rhinocerus Cooke, were characterized to be a polysaccharide-protein complex and a glucan, respectively. Their in vitro antitumor activities against different leukemic cell lines were investigated. PR-HW demonstrated significant growth inhibition of human acute promyelocytic leukemic cells (HL-60), chronic myelogenous leukemia cells (K562), and human acute monocytic leukemia cells (THP-1) in vitro, whereas PR-CA showed no such inhibition. Flow cytometric analysis has shown that the antiproliferative effect of PR-HW on HL-60 was mediated by cell cycle arrest at the G, phase, which subsequently led to apoptosis. This suggests that structural differences in PR-HW and PR-CA greatly affects their antitumor activities.