Intestinal mucosal barrier aging is a major cause of the occurrence and development of many chronic diseases in older adults and is closely associated with gut microbiota. However, it remains unclear whether exogenous supplementation with omega-3 polyunsaturated fatty acids (ω-3 PUFAs) can ameliorate aging-induced intestinal mucosal barrier damage by regulating the gut microbiota. This study was conducted to explore the roles of ω-3 PUFAs and gut microbiota in the process of maintaining the intestinal mucosal barrier. Senescence-accelerated mouse prone 8 (SAMP8) mice were used to establish a geriatric animal model and given reasonable doses of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) supplemented diets for six months to validate the effect of ω-3 PUFAs. Differences in the composition and function of gut microbiota were analyzed using 16S rRNA gene sequencing. This study revealed that aging SAMP8 mice showed increased intestinal permeability and gut microbiota disorder. Long-term supplementation with ω-3 PUFAs reduced intestinal mucosal permeability (P < 0.05), regulated gut microbiota, strengthened the microecological interactions, and enhanced enrichment of metabolic pathways producing short-chain fatty acids (P < 0.05), ultimately ameliorating intestinal mucosal barrier dysfunction. Furthermore, we demonstrated that ω-3 PUFAs could maintain the aging intestinal mucosal barrier by regulating the relative abundance of eight key characteristic microbial strains to increase the concentration of DHA and EPA in the plasma and colon tissue and reduced intestinal permeability of SAMP8 mice. ω-3 PUFAs can significantly ameliorate intestinal mucosal barrier function in senescence-accelerated mice, and this effect is mediated by the gut microbiota. The findings of our study provide a scientific basis for the rational intake of ω-3 PUFAs in older adults to maintain intestinal mucosal barrier function.