In this study, five novel polysaccharides SLNT1, SLNT2, JLNT1, JLNT2, and JLNT3 were isolated from the fruit body of Lentinus edodes. Chemical and physical analyses showed that the five polysaccharides consist of glucose with the structure of β-(1→3)-d-glucose main chains and β-(1→6)-d-glucose side chains. Moreover, all of them had triple-helical conformation and different molecular weight distributions. Animal studies further demonstrated that the antitumor effects were remarkably improved by SLNT1 and JLNT1 treatments with the inhibitory rates of 65.41% and 61.07% in H22-bearing mice, respectively. Additionally, both of them significantly increased the levels of serum IL-2 and TNF-α production and induced the tumor cell apoptosis. Taken together, our findings revealed that the involved antitumor mechanisms possibly in part were mediated not only by enhancing the immunity but also by directly killing the tumor and the induction of tumor cell apoptosis in H22-bearing mice.