The incidence of inflammatory bowel disease (IBD) has continued to increase worldwide, and a caspase-in-dependent, proinflammatory form of programmed cell death termed necroptosis has been observed to actively play an important role in its pathogenesis. Recent studies have indicated that polysaccharides from edible mushrooms suppress colitis. However, there is a lack of information on the effect of mushroom polysaccharides on colitis-associated necroptosis. In this study, the anti-inflammatory activity of polysaccharides from Lentinus edodes and their impact on colitis-associated necroptosis was investigated using both in vivo and in vitro models. Polysaccharides extracted from L. edodes were administered to mice with dextran sodium sulphate-induced colitis prior to and during colitis induction. The Caco-2 cell model of necroptosis was used to investigate the antinecroptotic activity of the polysaccharide sample in an in vitro system. We found that polysaccharides from L. edodes suppressed colitis in mice in a dose-dependent manner and inhibited necroptotic cell death in Caco-2 cells. Interestingly, the polysaccharide extract exerted a remarkable inhibitory effect on the receptor-interacting protein kinase RIPK1-RIPK3-MLKL (mixed lineage kinase domain-like pseudokinase) necroptosis signaling cascade, which resulted in a decreased level of phosphorylated MLKL in the colon of mice with colitis. Notably, the anti-inflammatory and antinecroptotic activities of the polysaccharide sample were found to be dependent on the carbohydrate-rich fraction of the polysaccharides. These results suggested that the inhibitory effect of the polysaccharides from L. edodes on necroptotic cell death in the colon may be partly responsible for its anti-inflammatory activity against ulcerative colitis. Therefore, this study provides evidence for the antinecroptotic and anti-inflammatory activity of L. edodes polysaccharide extract to support its use as an alternative source of therapeutic agent against ulcerative colitis.