Recurrent and intractable colitis is very distressing for patients. Natural dietary fibers and polysaccharides have shown great potential in the prevention and management of Inflammatory bowel disease. This study aimed to investigate the ameliorative effects of arabinoxylan (AX), derived from barley bran, on dextran sulfate sodium induced ulcerative colitis in murine models, while also elucidating its underlying mechanisms. Additionally, the combined therapeutic effects of AX and 5-Aminosalicylic acid (5-ASA) were evaluated. The results indicated that AX significantly improved colonic inflammation, including restored body weight, colon length, reduced disease activity index (DAI) scores, and histopathological scores. AX intervention significantly reduced colonic inflammation and oxidative stress levels, while enhancing the expression of tight junction proteins. Furthermore, AX exhibited excellent performance in restoring intestinal mucus barrier function, by up-regulated the expression of MUC-2 and proteins associated with goblet cell secretion. Notably, AX selectively enriched beneficial bacterial species, such as Ligilactobacillus, while significantly reducing the abundance of Escherichia-Shigella, improving microbiota composition, and increasing short-chain fatty acids (SCFAs) levels in the colon. Moreover, the combination therapy of AX and 5-ASA compensated for the limitations of 5-ASA monotherapy, particularly in promoting intestinal mucosal barrier repair and enhancing SCFAs production, thereby mitigating colitis symptoms through multiple mechanisms.