We have recently shown that age-dependent regional brain atrophy and lateral ventricle expansion may be linked with impaired cognitive and locomotor functions. However, metabolic profile transformation in different brain regions during aging is unknown. This study examined metabolic changes in the hippocampus, medial prefrontal cortex (mPFC) and striatum of middle- and late-aged Sprague-Dawley rats using ultrahigh performance liquid chromatography coupled with high-resolution accurate mass-orbitrap tandem mass spectrometry. Thirty-eight potential metabolites were altered in hippocampus, 29 in mPFC, and 14 in striatum. These alterations indicated that regional metabolic mechanisms in lated-aged rats are related to multiple pathways including glutathione, sphingolipid, tyrosine, and purine metabolism. Thus, our findings might be useful for understanding the complexity of metabolic mechanisms in aging and provide insight for aging and health span.