Objective This study aimed to investigate serum zonulin and claudin-5 levels of children and adolescents with attention-deficit/hyperactivity disorder (ADHD) and healthy controls by controlling the parameters such as age, sex and body mass index (BMI) percentile which are known to affect these parameters. Method A total of 80 treatment-naive children and adolescents with ADHD and 40 healthy volunteer controls aged 8-12 years were enrolled in this study. The severities of ADHD symptoms were assessed via parent- and teacher-rated questionnaires. The severity of anxiety and depression symptoms of the children were assessed by the self-report inventories. Serum levels of zonulin and claudin-5 were measured using commercial enzyme-linked immunosorbent assay kits. Results The multivariate analysis of covariance (MANCOVA) revealed a significant main effect of groups in the serum zonulin and claudin-5 levels, an effect that was independent of age, sex and BMI percentile. Significant differences were found between the study groups in terms of serum log-claudin-5 levels. However, there was no significant difference between the study groups in terms of serum zonulin levels Conclusion These findings provide additional evidence for dysregulation of the blood-brain barrier, especially abnormalities in claudin-5 function, which may be involved in the aetiology of ADHD.