Calorie restriction (CR) promotes healthy aging and lifespan extension, but how the apolipoprotein E (APOE) genotype, a genetic polymorphism associated with longevity and Alzheimer's disease, modulates its effects remains unclear. Using humanized APOE mice, we found that CR decreased metabolic rates, neuroinflammation and anxiety, and improved associative memory, in aged APOE3 (E3) and APOE4 (E4) mice, but not APOE2 (E2) mice. CR upregulated cholesterol synthesis pathways in the brains of E3 and E4 mice, enhancing oligodendrocyte precursor cell differentiation and myelination, with minimal effects in E2 mice. Lipidomics revealed limited changes in brain lipids across genotypes but significant alterations in serum lipids in E2 mice. Additionally, CR promoted a healthier gut microbiota in E3 and E4 mice but had opposing effects in E2 mice. These findings highlight APOE genotype-specific responses to CR across multiple dimensions, providing valuable insights for personalized strategies for aging prevention and the treatment of aging-related diseases.