Grifola frondosa antioxidant peptides (GFAP) were prepared through trypsin enzymolysis and characterized. This study conducted a comprehensive assessment of clinical symptoms, colon pathological injuries, levels of inflammatory factors, expression of inflammation-related proteins, and alterations in gut microbiota composition in mice with ulcerative colitis (UC). The findings demonstrated that GFAP effectively mitigated UC, alleviated mucosal damage, and reduced inflammatory infiltration. Specifically, GFAP administration resulted in significant reductions in pro-inflammatory cytokines IL-6, IL-1β, and TNF-α, while enhancing the expression levels of tight junction proteins such as Occludin and ZO-1. Additionally, GFAP treatment led to decreased levels of Toll-like receptor 4 (TLR-4), inducible nitric oxide synthase (iNOS), and TNF-α. Noteworthy, GFAP also influenced the gut microbiota by decreasing the abundance of Proteobacteria and increasing Bacteroidetes and Firmicutes. Moreover, specific bacteria like Bacteroides uniformis and Alistipes exhibited elevated abundances following GFAP treatment. In summary, GFAP exhibited preventive and protective effects against UC in mice by effectively alleviating clinical symptoms and modulating gut microbiota composition.