Hypoxic pulmonary vasoconstriction (HPV) is a physiological vasomotor response that maintains systemic oxygenation by matching perfusion to ventilation during alveolar hypoxia. Although mitochondria appear to play an essential role in HPV, the impact of mitochondrial dysfunction on HPV remains incompletely defined. Mice lacking the mitochondrial complex I (CI) subunit Ndufs4 ( Ndufs4^−/−) develop a fatal progressive encephalopathy and serve as a model for Leigh syndrome, the most common mitochondrial disease in children. Breathing normobaric 11% O₂ prevents neurological disease and improves survival in Ndufs4^−/− mice. In this study, we found that either genetic Ndufs4 deficiency or pharmacological inhibition of CI using piericidin A impaired the ability of left mainstem bronchus occlusion (LMBO) to induce HPV. In mice breathing air, the partial pressure of arterial oxygen during LMBO was lower in Ndufs4^−/− and in piericidin A-treated Ndufs4^+/+ mice than in respective controls. Impairment of HPV in Ndufs4^−/− mice was not a result of nonspecific dysfunction of the pulmonary vascular contractile apparatus or pulmonary inflammation. In Ndufs4-deficient mice, 3 wk of breathing 11% O₂ restored HPV in response to LMBO. When compared with Ndufs4^−/− mice breathing air, chronic hypoxia improved systemic oxygenation during LMBO. The results of this study show that, when breathing air, mice with a congenital Ndufs4 deficiency or chemically inhibited CI function have impaired HPV. Our study raises the possibility that patients with inborn errors of mitochondrial function may also have defects in HPV.