This study was conducted to investigate the prebiotic potential of Chlorella pyrenoidosa polysaccharides to provide useful information for developing C. pyrenoidosa as a green healthy food. C. pyrenoidosa polysaccharides were prepared and their physicochemical characteristics were determined. The digestibility and fermentation characteristics of C. pyrenoidosa polysaccharides were evaluated using in vitro models. The results revealed that C. pyrenoidosa polysaccharides were composed of five non-starch polysaccharide fractions with monosaccharide compositions of Man, Rib, Rha, GlcA, Glc, Gal, Xyl and Ara. C. pyrenoidosa polysaccharides could not be degraded under saliva and the gastrointestinal conditions. However, the molecular weight and contents of residual carbohydrates and reducing sugars of C. pyrenoidosa polysaccharides were significantly reduced after fecal fermentation at a moderate speed. Notably, C. pyrenoidosa polysaccharides could remarkably modulate gut microbiota, including the promotion of beneficial bacteria, inhibition of growth of harmful bacteria, and reduction of the ratio of Firmicutes to Bacteroidetes. Intriguingly, C. pyrenoidosa polysaccharides can promote growth of Parabacteroides distasonis and increase short-chain fatty acid contents, thereby probably contributing to the promotion of intestinal health and prevention of diseases. Thus, these results suggested that C. pyrenoidosa polysaccharides had prebiotic functions with different fermentation characteristics compared with conventional prebiotics such as fructooligosaccharide, and they may be a new prebiotic for improving human health.