Abstract: Objective: To evaluate the therapeutic potential of human milk oligosaccharides (HMOs) in mitigating age-related intestinal barrier dysfunction using D-galactose-induced aging mouse model. Methods: Aging was induced in mice via chronic intraperitoneal administration of D-galactose (450 mg/kg body weight BW) for 8 weeks. Concurrently, mice were orally administered HMOs—2'-fucosyllactose (2'-FL), 3'-sialyllactose (3'-SL), and lacto-N-neotetraose (LNnT)—at a dose of 500 mg/kg BW daily. After 8 weeks, the body weight changes, intestinal organ indices, oxidative stress levels, intestinal permeability, colonic morphology, and the proliferation and differentiation capabilities of stem cells in mice were measured and analyzed. Results: Following HMOs intervention, aging model mice demonstrated a pronounced recovery of intestinal health, with organ indices exhibiting a significant increase of 27.16%~33.74%（P<0.05）. Serum superoxide dismutase content increased significantly by 34.82% (P<0.001), while malondialdehyde content decreased by 46.21%~61.79% (P<0.05 or P<0.01). Intestinal barrier integrity was reinforced, with FITC-dextran permeability decreasing by 48.45~62.84% (P<0.05 or P<0.01). Histological analyses revealed remarkable regenerative effects on colonic architecture, including significant increases in crypt number, crypt depth, proliferating cell nuclear antigen (PCNA)-positive cell counts, and cell differentiation (P<0.05或P<0.01). Conclusion: HMOs intervention, particularly with 2'-FL, significantly enhanced the proliferation and differentiation capabilities of intestinal stem cells in D-galactose-induced aging mice. It improved the atrophy of colonic crypt morphology, strengthened the intestinal mucus barrier, and reduced intestinal permeability, thereby ameliorating the aging intestinal barrier.