Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disease for which specific biomarkers and pathological mechanisms have yet to be identified. Methylphenidate (MPH) is commonly used to treat ADHD, but its therapeutic mechanisms and its impact on brain metabolites remain unclear. Metabolomics can help to discover biomarkers and identify pathophysiological mechanisms. We adopted an untargeted metabolomics approach based on gas chromatography-mass spectrometry to investigate the potential biomarkers and pathogenesis of ADHD. Ten Wistar-Kyoto (WKY) rats were chosen as healthy controls (vehicle, i.g.). Twenty young spontaneously hypertensive rats (SHR) were randomly allocated to the SHR group (vehicle, i.g.) and MPH group (2 mg/kg/day, i.g.). We identified 103 metabolites from the prefrontal cortex (PFC). Orthogonal partial least square-discriminate analysis showed the differential expression of these metabolites between the groups. Multivariate and univariate statistical analyses isolated 12 metabolites that differed significantly between the WKY and SHR groups: 3-hydroxymethylglutaric acid, 3-phosphoglyceric acid, adenosine monophosphate, cholesterol, lanosterol, and o-phosphoethanolamine; 3-hydroxymethylglutaric acid and cholesterol were reversed with MPH treatment. Pathway and enrichment analyses revealed that the altered metabolites belonged to the cholesterol metabolism pathways. ELISA and western blotting showed that the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase and the expression of sterol regulatory element-binding protein-2 and ATP-binding cassette transporter A1 were reduced in the PFC of the SHR; the latter two proteins were upregulated by MPH. In conclusion, metabolomics analysis identified potential biomarkers that influence cholesterol metabolism and may be implicated in the development of ADHD-like behavior. MPH can regulate cholesterol metabolism in the PFC of ADHD models. This study uncovered potential biomarkers and pathways involved in ADHD, providing new insight into its pathogenesis. Copyright © 2019 Elsevier Inc. All rights reserved.