Impaired vasodilation in persistent pulmonary hypertension of the newborn (PPHN) is characterized by mitochondrial dysfunction. We investigated the hypothesis that decreased nitric oxide (NO) availability leads to impaired mitochondrial biogenesis and function in a lamb model of PPHN induced by prenatal ductus arteriosus constriction. We ventilated PPHN lambs with 100% O₂ alone or with inhaled nitric oxide (iNO). We treated pulmonary artery endothelial cells (PAEC) from control and PPHN lambs with detaNONOate, an NO donor. We observed decreased mitochondrial (mt) DNA copy number, electron transport chain (ETC) complex subunit levels and ATP levels in PAEC and lung tissue of PPHN fetal lambs at baseline compared to gestation matched controls. Phosphorylation of AMP-activated kinase (AMPK) and levels of PPAR gamma coactivator 1-alpha (PGC-1α) and sirtuin1, which facilitate mitochondrial biogenesis, were decreased in PPHN. Ventilation with 100% O₂ was associated with larger decreases in ETC subunits in the lungs of PPHN lambs compared to unventilated PPHN lambs. INO administration, with weaning of FiO₂ partly restored mtDNA copy number, ETC subunit levels and ATP levels. DetaNONoate increased eNOS phosphorylation and its interaction with HSP90, levels of superoxide dismutase 2 (SOD2) mRNA, protein and activity and decreased the mitochondrial superoxide in PPHN-PAEC. Knockdown of eNOS decreased ETC proteins in control PAEC, while detaNONOate increased ETC protein levels and ATP-linked O₂ consumption in eNOS knockdown cells and in PPHN-PAEC. We conclude that ventilation with 100% O₂ amplifies oxidative stress and mitochondrial dysfunction in PPHN, which are partly improved by supplemental NO and weaning of oxygen.