In the process of aging, adverse changes such as weakened intestinal barrier function, increased chronic inflammation, and decreased gut microbiota diversity often occur. We explored the protective effects of Oat β-glucan (BG) on the gut homeostasis of naturally aging mice. The study shows that daily intervention with 400 mg/kg BG effectively modulates the intestinal mucosal structure, mechanical barrier function [Zonula occludens-1 (ZO-1), occludin, and claudin], and anti-inflammatory [Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6), and IL-1β], as well as antioxidant responses in aging mice. Spearman correlation analyses showed that BG supplementation increased acetate levels by 1.8-fold, propionate levels by 2.5-fold, and butyrate-derived GABA levels by 2.5-fold. Additionally, BG supplementation improved the gut microbiota, increasing the abundance of beneficial bacteria like Bacteroidota, Prevotellaceae, Coprobacillaceae, and Faecalibacterium. These microbes metabolize BG to produce short-chain fatty acids (SCFAs), activating butanoate and propanoate metabolic pathways to maintain intestinal homeostasis. In conclusion, this study identifies the therapeutic effects of BG in regulating intestinal barrier homeostasis and gut microbiota, providing new insights for nutritional intervention strategies in the elderly.