The aging brain shows changes in microglial function, morphology, and phenotype, indicating chronic microglial activation. CX3CR1 is crucial for microglial chemotaxis, phagocytosis, and activation. However, its exact role in the aging brain is not well understood. In this study, we examined the expression of CX3CR1 in the brains of middle-aged mice (10 months old) and explored its functional implications by conducting proteomic profiling in CX3CR1-deficient mouse cerebrum. Proteomic analysis revealed an enrichment of differentially expressed proteins (DE-proteins) in the cerebrum of middle-aged mice in GO pathways such as “synapse”, “translation”, and “ribosome”. CX3CR1 deficiency affected protein levels in GO pathways such as “glutamatergic synapse” and “RNA splicing.” We also detected the proteomics in age-matched 5xFAD mice for comparison, since this mouse strain featured aberrant microglial activation and CX3CR1 upregulation in the hippocampus and frontal cortex. Our findings demonstrated that CX3CR1 was upregulated to maintain synaptic homeostasis probably through regulating microglial activation and phagocytosis in the brains of middle-aged mice. CX3CR1 may represent a promising therapeutic target for alleviating the effects of aging and preventing neurodegeneration.