The seeds from Gleditsia sinensis Lam., a common ecologically and economically useful tree, have high economic and nutritional value. The protective effect of polysaccharides from Gleditsia sinensis Lam. seeds (ZJMP) against dextran sulfate sodium-induced colitis in mice was investigated in this study. ZJMP alleviated weight loss, reduced the disease activity index, prevented colon shortening, alleviated colonic tissue damage, and restored goblet cell secretion in colitic mice. Dietary ZJMP reduced proinflammatory cytokine overproduction in the colonic mucosa and serum, which was accompanied by suppression of NO levels and MPO and SOD activities. The addition of ZJMP increased the expression of Muc2 and tight junction proteins. Furthermore, dietary ZJMP partially reversed the alteration of gut microbiota in colitic mice by boosting the abundance of beneficial bacteria like Akkermansia, Lactobacillus, and Christensenella while lowering the abundance of harmful bacteria like Bacteroides, Prevotella, and Mucispirillum. Additionally, the decreased production of short-chain fatty acids in the colitic mice was recovered by ZJMP administration. The findings demonstrated the anti-inflammatory properties and mechanism of dietary ZJMP in the colon, which is essential for the sensible application of ZJMP in the prevention and amelioration of inflammation-related diseases as a nutritional supplement.