This study aimed to investigate the therapeutic efficacy of Larimichthys Crocea Isinglass polysaccharide (CIP) in ameliorating DSS-induced colitis. CIP, a complex polysaccharide composed of six monosaccharides with a molecular weight of 3.97 kDa. Specifically, CIP treatment mitigated weight loss and histopathological damage in the colon, increased colon length, and by inhibiting the TLR4/MyD88/NF-κB pathway significantly downregulated the secretion levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, while simultaneously upregulating the anti-inflammatory cytokine IL-10 and inhibition of the oxidative stress-related enzyme inducible nitric oxide synthase (iNOS). Additionally, CIP treatment enhanced intestinal barrier function by increasing the expression of intestinal tight junction proteins (TJs) and MUC2, and it promoted the production of short-chain fatty acids (SCFAs). Concurrently, CIP modulated the composition of the intestinal microbiota, inhibiting the proliferation of potentially harmful bacteria such as Bacteroidota and Campylobacter. Taken together, these findings suggest that CIP exhibits promising attributes as a functional food constituent, with a particular efficacy in modulating and maintaining intestinal health.