This study aimed to investigate the possible mechanism of the protective effect of a sulfated polysaccharide (SP) from Gracilaria Lemaneiformis against colitis induced by dextran sulfate sodium (DSS). Balb/c mice were gavaged with SP for four weeks, then colon tissue, cecal contents and feces were collected for further analysis. Results showed that SP was effective for inhibiting colon shortening and oedema forming. It could alleviate colonic inflammation via down-regulating the expression of tumor necrosis factor-α (TNF-α), interleukin (IL-6, IL-1β). Besides, it enhanced the intestinal barrier by up-regulating the expression of tight junction proteins Claudin-1 and Zonula occludens-1 (ZO-1) as well as Mucin (MUC-2). The increased expression of short chain fatty acid (SCFA) receptors including G protein-coupled receptor (GPR43, GPR109A) and olfactory receptor (Olfr78), and SCFA production in feces indicated that most of SCFA were absorbed in colon, which could play positive roles in ameliorating colitis. Furthermore, the results of gut microbiota showed that Enterorhabdus, Desulfovibrio, Alistipes, Bacteroides acidifaciens had closest correlations with the strongest protective effects against colitis. Therefore, SP could alleviate DSS-induced colitis via enhancing intestinal barrier, reducing inflammation, activating SCFA receptors and regulating gut microbiota. It could be developed as functional foods which is good for gut health.