Introduction Human herpesvirus 6 (HHV-6) is the second most common causative pathogen of acute encephalopathy in immunocompetent children in Japan. Identification of biomarkers associated the pathophysiology is desirable to monitor disease severity, progression, and prognosis.Objectives To investigate potential biomarkers for HHV-6 encephalopathy, serum metabolome profiling was analyzed and candidate metabolites were investigated the function in the diseases.Methods Pediatric patients with HHV-6 encephalopathy (n = 8), febrile seizure (n = 20), and febrile infection without febrile seizure (n = 7) were enrolled in this study, and serum metabolites were identified and quantified. For further analysis, serum samples of HHV-6 infected patients were analyzed by absolute quantification assay for kynurenine (KYN) and quinolinic acid (QUIN) in a total of 38 patients with or without encephalopathy. An in vitro blood-brain barrier (BBB) model was used to evaluate the effect of KYN and QUIN on BBB integrity because BBB damage induces brain edema.Results Metabolome profiling identified 159 metabolites in serum samples. The levels of KYN and QUIN, which belong to the tryptophan-KYN pathway, were significantly higher in the HHV-6 encephalopathy group than the other two groups. When quantified in the larger patient group, remarkably high levels of KYN and QUIN were observed exclusively in the encephalopathy group. Trans-endothelial electrical resistance of the BBB model was significantly decreased after QUIN treatment in culture.Conclusion Metabolome analysis revealed that KYN and QUIN may be associated with the pathophysiology of HHV-6 encephalopathy. In particular, QUIN may damage BBB integrity.