In recent years, an increasing number of studies have used metabolomics techniques to uncover the underlying mechanisms of diseases, including substance abuse. Although physical exercise has been considered as a potential treatment for methamphetamine (MA) abuse, there is still a lack of studies regarding exercise modulating the metabolic profile of the brain following MA use. This untargeted metabolomics study, based on liquid chromatography-mass spectrometry (LC-MS), investigated the effects of aerobic exercise on MA-induced metabolism in mice brain. We found that 2 weeks of treadmill training modulates MA-induced metabolic disturbances in the brain. Among these metabolites, 129 metabolites with significant differences were identified under MA induction, and 32 metabolites were significantly affected by exercise. The relevant differential metabolites were mainly enriched in glycerophospholipid metabolism, steroid hormone biosynthesis and degradation, and renin-angiotensin system pathways. To our knowledge, this is the first study to use LC-MS approach to explore the effects of aerobic exercise on MA-induced metabolic profiles in the brain. The findings of this study provide new insights into exercise therapy for MA use.