As potential candidates for treating ulcerative colitis (UC), polysaccharides have been attracting extensive interest in recent years. Cordyceps sinensis (C. sinensis) is a kind of traditional Chinese edible food, and its polysaccharide fractions have been found to be effective in regulating immunity and protecting the kidneys. To determine the potential function of polysaccharides from natural C. sinensis on UC, their effects in terms of histological, serological, biochemical, and immunological aspects on dextran sulphate sodium (DSS)-induced colitis mice model were investigated. Results showed that the polysaccharides significantly alleviated colitis by increasing the colon length, alleviating colon tissue damage, and inhibiting the activation of the NF-κB pathway. In addition, polysaccharides reduced the contents of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the serum, increased the number of goblet cells, and improved the expression of intestinal tight junction proteins (Occludin and Claudin-1). They also evidently enhanced the formation of IgA-secretory cells and sIgA contents. Furthermore, the polysaccharides modulated the gut microbiota by decreasing the relative abundance of Bilophila and increasing the relative abundance of Dehalobacterium, Coprococcus, Oscillospira, and Desulfovibrio, which is accompanied by an increase in the short chain fatty acids’ (SCFAs) concentrations in cecal contents. These results suggested that C. sinensis polysaccharides possessed promising intervening effects on experimental acute UC in mice.