Ulcerative colitis (UC) is frequently accompanied by inflammation, oxidative stress and intestinal barrier injury. To investigate the protective effects and underlying mechanisms of Laminaria japonica fucoidan (LJF) against UC, three in vitro cell models were constructed to evaluate the anti-UC activity of LJF. Results showed that, compared with commercial fucoidan (CF), LJF exhibited higher contents of uronic acid and fucose, a higher molecular weight, and a more intact and compact microstructure. Within the concentration range of 12.5–200 μg·mL-1, LJF exhibited a dose?dependent increase in efficacy. In the RAW264.7 inflammatory cell model, the levels of inflammatory mediators were downregulated and anti?inflammatory cytokines were upregulated by LJF, and the underlying mechanism may involve the inhibition of the Toll-like receptor 2/nuclear factor-κB/p38 mitogen-activated protein kinase pathway and the expression of the inflammasome. Furthermore, 200 μg·mL-1 LJF regulated the expression of oxidative stress-related genes and tight junction protein genes in oxidatively damaged Caco-2 cells, reducing intracellular reactive oxygen species by 68.98% and elevating cell viability by 67.70% compared with the model group. Additionally, relative to the model group, the disruption of the Caco?2 monolayer barrier was ameliorated by LJF, leading to a 19.94% increase in cell viability and a 65.06% increase in transepithelial electrical resistance values. In vivo results demonstrated that oral administration of LJF alleviated macroscopic symptoms. In terms of in vitro and in vivo experiments, LJF demonstrated superior efficacy to commercial fucoidan and effects comparable to those of mesalazine. These findings indicate that the anti-UC activity of LJF is exerted through multiple pathways including anti-inflammation, antioxidation, and protection of the intestinal epithelial barrier, providing a theoretical basis for nutritional intervention for UC and the development of related functional foods.