Phenylketonuria (PKU) is a disorder of phenylalanine (Phe) metabolism caused by impaired phenylalanine hydroxylase activity, resulting in increased levels of Phe in fluids and tissues of patients. PKU patients present severe intellectual deficit. The aim of this study was to analyze brain bioenergetics in the pathophysiology of hyperphenylalaninemia (HPA). For this study, 30-day-old Wistar rats were randomized into two groups: HPA group received a single subcutaneous administration of Phe (5.2 μmol/g) plus p-Cl-Phe (PAH inhibitor) (0.9 μmol/g), and control group received a single injection of NaCl 0.9%. One hour after, animals were euthanatized and the cerebral cortex, hippocampus and striatum were collected. The activities of important bioenergetics enzymes activities, concentrations of glycogen, phosphate levels, as well as mRNA expression mitochondrial biogenesis and dynamics markers were evaluated. In cerebral cortex, HPA group showed decreased CK activity, glycogen levels, complex I-III, IV and V activities, as well as PDH and CS activities, as well as increased LDH, α-KGDH and IDH activities, and the levels of free and total Pi. In striatum, HPA animals presented increased LDH and IDH activities, and decreased α-KGDH and complex IV activities. In hippocampus, HPA rats had increased α-KGDH and IDH activities, decreased complexes I and IV activities, and increased mRNA expression of MFN1. Our data demonstrated impaired bioenergetics in cerebral cortex, striatum and hippocampus of HPA rats. In conclusion, it may be speculated that disruption of brain bioenergetics is involved in neuropathology seen in PKU patients.