3-Hydroxy-3-methylglutaryl-CoA lyase (HL) deficiency is a neurometabolic disorder characterized by predominant accumulation of 3-hydroxy-3-methylglutaric acid (HMG) in tissues and biological fluids. Patients often present in the first year of life with metabolic acidosis, non-ketotic hypoglycemia, hypotonia, lethargy, and coma. Since neurological symptoms may be triggered or worsened during episodes of metabolic decompensation, which are characterized by high urinary excretion of organic acids, this study investigated the effects of HMG intracerebroventricular administration on redox homeostasis, citric acid cycle enzyme activities, dynamics (mitochondrial fusion and fission), and endoplasmic reticulum (ER)-mitochondria crosstalk in the brain of neonatal rats euthanized 1 (short term) or 20 days (long term) after injection. HMG induced lipid peroxidation and decreased the activities of glutathione peroxidase (GPx) and citric acid cycle enzymes, suggesting bioenergetic and redox disruption, 1 day after administration. Levels of VDAC1, Grp75, and mitofusin-1, proteins involved in ER-mitochondria crosstalk and mitochondrial fusion, were increased by HMG. Furthermore, HMG diminished synaptophysin levels and tau phosphorylation, and increased active caspase-3 content, indicative of cell damage. Finally, HMG decreased GPx activity and synaptophysin levels, and changed MAPK phosphorylation 20 days after injection, suggesting that long-term toxicity is further induced by this organic acid. Taken together, these data show that HMG induces oxidative stress and disrupts bioenergetics, dynamics, ER-mitochondria communication, and signaling pathways in the brain of rats soon after birth. It may be presumed that these mechanisms underlie the onset and progression of symptoms during decompensation occurring in HL-deficient patients during the neonatal period.