Rationale <p>Autism spectrum disorders (ASD) are a group of neurodevelopmental and multifactorial conditions with cognitive manifestations. The valproic acid (VPA) rat model is a well-validated model that successfully reproduces the behavioral and neuroanatomical alterations of ASD. Previous studies found atypical brain connectivity and metabolic patterns in VPA animals: local glucose hypermetabolism in the prefrontal cortex, with no metabolic changes in the hippocampus.</p> Aim <p>This study aimed to explore mitochondrial structural features, lipid content, and functionality in the hippocampus and cerebral cortex in the VPA model.</p> Methods <p>On embryonic day 10.5, pregnant Wistar rats were injected with VPA (450&#xa0;mg/kg) or saline solution. In the hippocampus and cerebral cortex of male offspring (postnatal day 35), the mitochondrial structure was evaluated by transmission electron microscopy, oxidized/reduced glutathione was determined by high-performance liquid chromatography, mitochondrial membrane cholesterol, and phospholipids were determined by thin-layer chromatography, and oxygen consumption and ATP synthesis were measured in isolated mitochondria.</p> Results <p>Mitochondrial increased oxygen consumption and decreased ATP production, increased oxidized/reduced glutathione, cholesterol accumulation in mitochondrial membrane and altered mitochondrial structure were found in the hippocampus of VPA animals. All parameters were preserved in the cerebral cortex of VPA rats.</p> Conclusions <p>These findings reveal brain region-specific mitochondrial structural and functional alterations in VPA-treated animals, with preserved mitochondria in regions with high glucose demand and impaired mitochondria in metabolically normal areas. Moreover, cholesterol accumulation in hippocampal mitochondrial membranes is a potential cause of mitochondrial dysfunction, contributing to a prooxidant state.</p>