To discern whether tuna oil modulates the expression of brain proteins and the gut microbiota structure during aging induced by D-galactose, we generated an aging mouse model with D-galactose treatment, and the mice showed aging and memory deterioration symptoms according to physiological and biochemical indices. Treatment with different doses of tuna oil alleviated the symptoms, the high dose showed a better effect. Subsequently, brain proteomic analysis showed the differentially expressed proteins were involved in damaged synaptic system repairment and signal transduction system enhancement. In addition, tuna oil treatment restored the diversity of gut microbiota, 27 key OTUs, which were identified using a redundancy analysis, were significantly correlated with at least one physiological index and three proteins or genes. These findings suggest that the combination of proteomics and gut microbiota is an effective strategy to gain novel insights regarding the effect of tuna oil treatment on the microbiota-gut-brain axis.