Exercise is widely recognized for improving physical functions, learning, and memory. However, the mechanisms behind these effects are not fully understood. This study aims to investigate the potential mechanisms through which exercise enhances learning and memory in mice using multi-omics analysis. Twenty male C57BL/6J mice were divided into exercise and control groups. The exercise group underwent a 4-month treadmill training regimen (12 m/min). Learning and memory abilities were assessed using the Morris water maze test. Brain, serum, and fecal samples were collected for neurotransmitter analysis, serum metabolomics analysis, and gut microbiota analysis. Data from neurotransmitter and serum metabolomics analyses were integrated with gut microbiota analysis. For comparisons between the two groups, the independent sample t-test was employed. For comparisons involving multiple groups, a repeated measures one-way analysis of variance (ANOVA) with random unit group design was applied. Statistical significance was defined as P < 0.05. The Morris water maze test significantly improved learning and memory in the exercise group (P < 0.05). Neurotransmitter analysis revealed significant differences in cognitive function-related neurotransmitters and pathways between the exercise and control groups (P < 0.05). Serum metabolomics analysis identified differences in serum metabolites between the two groups, which were linked to key pathways involved in neural repair and cognitive function. Microbial sequencing showed greater gut microbiota diversity in the exercise group, with the most notable changes at the genus level, particularly in Allobaculum, A2, and Clostridium_sensu_stricto_1 (P < 0.05). Integrated analysis indicated correlations between changes in gut microbiota and serum metabolites associated with cognitive function. Long-term exercise enhances learning and memory in mice through multiple mechanisms, including neurotransmitter regulation, serum metabolite changes, and modulation of gut microbiota. These findings provide new insights into the neuroprotective effects of exercise.© 2025. The Author(s), under exclusive licence to Springer Nature B.V.