Tremella fuciformis is an edible and medicinal mushroom. Polysaccharides from T. fuciformis have received increasing attention due to their diversely pharmacological activities. In this study, the digestive behavior and fermentation characteristics of T. fuciformis polysaccharides (TFP) were studied. The results revealed that the reducing sugar content, chemical composition, molecular weight, rheological property, constituent monosaccharide, and FT-IR spectrum of TFP were not altered after the in vitro simulated digestion, indicating that it was indigestible under different simulated digestion conditions. However, the physicochemical characteristics of TFP, including reducing sugar content, molecular weight, constituent monosaccharide, and free monosaccharide released, were obviously altered after the in vitro fermentation for 48 h, indicating that it was remarkably utilized by intestinal microbiota in human feces. Notably, TFP could obviously modulate the microbial composition via promoting the relative abundances of Phascolarctobacterium, Bacteroides, and Lachnoclostridium. Moreover, TFP could also increase the production of short-chain fatty acids, including acetic, propionic, n-butyric, and n-valeric acids, after the in vitro fermentation for 48 h. These results showed that TFP was stable under the simulated digestion conditions, but could be utilized by intestinal microbiota in human feces, and might possess the potential to improve intestinal health.