Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF), a fatal disease associated with recurring lung infections due to ineffective clearance of the airways. As CF patients frequently suffer from ventilation-perfusion (VA/Q) mismatch, we speculated whether hypoxic pulmonary vasoconstriction (HPV), the physiological mechanism protecting from VA/Q inequalities, may be abrogated in this condition. Thus, functional CFTR may be critical for HPV. While the effects of CFTR in airway epithelial cells have been extensively studied, the functional role of CFTR in pulmonary arterial smooth muscle cells has thus far been largely obscure. In this study we characterized the role of CFTR in the pulmonary vasculature in acute hypoxia and linked our findings to the sphingolipid system, a known central mediator of HPV. In pulmonary artery smooth muscle cells (PASMCs), we studied the effects of CFTR deficiency or CFTR inhibition on two mechanisms known to be essential for the induction of HPV, (i) hypoxia-induced Ca2+ influx and (ii) hypoxia-induced translocation of transient receptor potential canonical 6 (TRPC6) to caveolae. In coimmunoprecipitation experiments in PASMCs, we further probed for direct protein-protein interaction between CFTR and TRPC6 upon hypoxia. VA/Q mismatch and HPV were studied in vivo or ex vivo in isolated perfused mouse lungs. Mice deficient in CFTR, sphingosine kinase (SphK) 1, sphingosine-1-phosphate (S1P) receptor 2 (S1P2) or S1P4 as well as the corresponding wild type mice were assessed. The effects of exogenous applied neutral sphingomyelinase (nSMase) on TRPC6 translocation and on the pulmonary arterial pressure were analyzed. Downstream signaling of nSMase and/or S1P was characterized. CFTR inhibition and/or deficiency reduced HPV ex vivo and led to aggravated hypoxemia following partial airway occlusion in vivo. Accordingly, CFTR inhibition diminished hypoxia-evoked Ca2+ signaling and TRPC6 translocation in PASMCs. Moreover, hypoxia led to a direct protein-protein interaction between CFTR and TRPC6, which was blocked by CFTR inhibition. Both, HPV and hypoxia-induced TRPC6 translocation were largely reduced by nSMase inhibition. Exogenous applied nSMase, however, induced TRPC6 translocation and mimicked the pulmonary vasopressor response evoked by hypoxia in a CFTR-dependent manner. Hypoxia- as well as nSMase-induced pulmonary vasoconstriction required S1P signaling as revealed by SphK inhibition and dual inhibition of S1P2 and S1P4. Analogously, HPV was reduced in lungs of SphK1-deficient mice as compared to wild type mice. Finally, the synergistic vasopressor response induced by nSMase and S1P was dependent on TRPC6, phospholipase C and rho kinase. Our data suggest a key role for CFTR in the induction of HPV. Thus, CFTR malfunction due to CFTR gene mutations may be causative for VA/Q mismatch in CF patients.