In this study, we evaluated the effects of tilapia head glycolipids (TH-GLs) on male C57BL/6 mice with inflammatory bowel disease (IBD) induced by dextran sulfate sodium (DSS) and the changes in gut microbiota compared with sulfasalazine. Mice were orally treated with 3% (w/v) DSS or not for 7 days, followed by drug treatment with TH-GLs or sulfasalazine. After treatment, macroscopic colitis symptoms, intestinal epithelial barrier function, inflammatory cytokines, and gut microbiota homeostasis were assessed. Further studies showed that TH-GLs and sulfasalazine showed different influences on the gut microbiota structure. Both sulfasalazine and TH-GLs decreased the DSS-induced enrichment of Gammaproteobacteria and Enterobacteriaceae. However, TH-GLs had a selective increase in the enrichment of Akkermansia, Prevotellaceae, Oscillospira, Allobaculum, Bifidobacterium, and Coprococcus in contrast to sulfasalazine, which selectively increased the enrichment of Dorea, Turicibacter, Bacteroides, Coprobacillus, Mucispirillum, and Dehalobacterium. In addition, both TH-GLs and sulfasalazine relieved body weight loss, and increased the immune organ index, while maintaining the balance between pro-inflammatory and anti-inflammatory cytokines. The results indicate that TH-GLs alleviate DSS-induced IBD in mice by decreasing the abundance of harmful gut microbiota and enhancing the abundance of probiotic gut microbiota. Thus, the mechanism through which TH-GLs inhibit inflammation through gut microbiota is different from that of sulfasalazine. Therefore, TH-GLs stand as potential prebiotics for the treatment of colonic inflammation and related diseases.