Aging, a complex biological process, is often accompanied by gut microbiota dysbiosis, metabolic pathway disorders, and cognitive dysfunction. In recent years, studies have shown that aerobic exercise can improve aging-related pathological phenotypes by modulating the gut-brain axis, but its molecular mechanisms have not been fully elucidated. In this study, using naturally aged mice as a model, we systematically integrated behavioral assessments and gut microbiota and metabolomics analysis to uncover molecular mechanisms upon aerobic exercise, reshaping a “microbiota-metabolite-brain” network to enhance the cognitive function of aged mice. Behavioral experiments showed that 12-week moderate-intensity treadmill training significantly enhanced spatial learning and memory capacity of aged mice, as evidenced by shortened escape latency in the Morris water maze and increased exploration efficiency in the target area. Histopathological analysis further confirmed that exercise intervention could ameliorate morphological abnormalities of hippocampal neurons, suggesting its protective effect against neurodegenerative diseases. Through 16S rRNA gene sequencing, we found that exercise intervention significantly increased gut microbiota diversity and specifically enriched beneficial bacteria such as Bifidobacterium, Parabacteroides, and Rikenella. Untargeted and targeted metabolomics analysis identified 32 differential metabolites involved in key signal pathways including energy metabolism reprogramming, antioxidant defense, and neuroprotection. Among them, L-homocitrulline and ADP-ribose associated with mitochondrial function, lithocholic acid with neuroinflammation-inhibiting function, and antioxidant L-carnosine presented an obvious increase. Microbiota-metabolite interaction network analysis showed a strongly positive correlation between the abundance of Parabacteroides and ADP-ribose, and a significant association between Bifidobacterium and lithocholic acid levels, suggesting that specific microbiota may mediate neuroprotective effects through metabolites. This study elucidated the specific mechanism upon aerobic exercise for enhancing brain function via the regulation of the gut microbiota-metabolite network: exercise-induced proliferation of beneficial bacteria can induce DNA repairing, inhibit neuroinflammation, and enhance antioxidant capacity through metabolic reprogramming, thereby synergistically rescuing impaired cognitive capacity. This finding provides a theoretical basis for targeted gut microbiota-based anti-aging strategies and the application potential of probiotics combined with personalized exercise intervention. Future studies can further combine gut microbiota transplantation and metabolite-targeted intervention to verify the cross-organ regulatory mechanisms of key metabolites and promote the transformation of synergistic intervention strategies in an "exercise-microbiota" mode towards precision medicine.