Jellyfish skin polysaccharides (JSP) were isolated from Rhopilema esculentum Kishinouye and contained 55.11% polysaccharides and 2.26% uronic acid. To examine the anti-inflammatory, antioxidant and immunomodulatory activities of JSP in vivo, C57BL/6 mice were induced to develop ulcerative colitis by dextran sulfate sodium (DSS) and the roles of dietary JSP supplementation in modulating colitis were explored. JSP supplementation reduced the symptoms of colitis in mice, increased colon length, protected goblet cells, and improved intestinal epithelial integrity and permeability. JSP modulated oxidative stress and inflammatory responses, which was demonstrated by reduced MPO activity, NO level, and levels of pro-inflammatory cytokines including TNF-α, IL-1β and IL-6 in mice. JSP suppressed NF-κB signaling pathways as evidenced by lower levels of phosphorylated p65 and IKB. Moreover, JSP supplementation enhanced the expression of tight junction proteins and mucins, and modulated the composition of the gut microbiota and the production of short-chain fatty acids. Taken together, these results reveal the anti-inflammatory effect of dietary JSP in vivo, suggesting the potential of JSP as a nutritional supplement or adjunct strategy in preventing or ameliorating colitis.