Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism to optimise lung gas exchange. We aimed to decipher the proposed oxygen sensing mechanism of mitochondria in HPV. Cytochrome redox state was assessed by remission spectrophotometry in intact lungs and isolated pulmonary artery smooth muscle cells (PASMC). Mitochondrial respiration was quantified by high-resolution respirometry. Alterations were compared with HPV and hypoxia-induced functional and molecular readouts on the cellular level. Aortic and renal arterial smooth muscle cells (ASMC and RASMC, respectively) served as controls. The hypoxia-induced decrease of mitochondrial respiration paralleled HPV in isolated lungs. In PASMC, reduction of respiration and mitochondrial cytochrome c and aa3 (complex IV), but not of cytochrome b (complex III) matched an increase in matrix superoxide levels as well as mitochondrial membrane hyperpolarisation with subsequent cytosolic calcium increase. In contrast to PASMC, RASMC displayed a lower decrease in respiration and no rise in superoxide, membrane potential or intracellular calcium. Pharmacological inhibition of mitochondria revealed analogous kinetics of cytochrome redox state and strength of HPV. Our data suggest inhibition of complex IV as an essential step in mitochondrial oxygen sensing of HPV. Concomitantly, increased superoxide release from complex III and mitochondrial membrane hyperpolarisation may initiate the cytosolic calcium increase underlying HPV.