The functional significance of the expression of cystic fibrosis transmembrane regulator (CFTR) on endothelial cells has not been elucidated yet. Since CFTR has been implicated in the regulation of intracellular sphingolipid levels which are important regulators of endothelial cell apoptosis in response to various insults, we investigated the role of CFTR in the apoptotic responses of lung endothelial cells. CFTR was detected as a functional chloride channel in primary lung endothelial cells isolated from both pulmonary arteries (human or mouse) and bronchial arteries (sheep). Both specific CFTR inhibition with DPC, CFTR_inh-172, or NPPB and CFTR knockdown significantly attenuated endothelial cell apoptosis induced by staurosporine or H₂O₂. CFTR_inh-172 treatment prevented the increases in the ceramide: sphingosine-1phosphate ratio induced by H₂O₂ in lung endothelial cells. Replenishing endogenous ceramides via sphingomyelinase supplementation restored the susceptibility of CFTR-inhibited lung endothelial cells to H₂O₂-induced apoptosis. Similarly, the anti-apoptotic phenotype of CFTR-inhibited cells was reversed by lowering the intracellular pH, and was reproduced by alkalinization prior to H₂O₂ challenge. TUNEL staining and active caspase-3 immunohistochemistry indicated that cellular apoptosis was decreased in lung explants from patients with cystic fibrosis compared to those with smoking-induced chronic obstructive lung disease, especially in the alveolar tissue and vascular endothelium. In conclusion, CFTR function is required for stress-induced apoptosis in lung endothelial cells by maintaining adequate intracellular acidification and ceramide activation. These results may have implications in the pathogenesis of cystic fibrosis, where aberrant endothelial cell death may dysregulate lung vascular homeostasis, contributing to abnormal angiogenesis and chronic inflammation.