Altered pulmonary angiogenesis contributes to disrupted alveolarization, which is the main characteristic of bronchopulmonary dysplasia (BPD). Transforming growth factor β (TGFβ) plays an important role during lung vascular development, and recent studies have demonstrated that endoglin is engaged in the modulation of TGFβ downstream signalling. Although there are two different isoforms of endoglin, L- and S-endoglin, little is known about the effect of S-endoglin in developing lungs. We analysed the expression of both L- and S-endoglin in the lung vasculature and its contribution to TGFβ-activin-like kinase (ALK)-Smad signalling with respect to BPD development. Hyperoxia impaired pulmonary angiogenesis accompanied by alveolar simplification in neonatal mouse lungs. S-endoglin, phosphorylated Smad2/3 and connective tissue growth factor levels were significantly increased in hyperoxia-exposed mice, while L-endoglin, phosphor-Smad1/5 and platelet-endothelial cell adhesion molecule-1 levels were significantly decreased. Hyperoxia suppressed the tubular growth of human pulmonary microvascular endothelial cells (ECs), and the selective inhibition of ALK5 signalling restored tubular growth. These results indicate that hyperoxia alters the balance in two isoforms of endoglin towards increased S-endoglin and that S-endoglin attenuates TGFβ-ALK1-Smad1/5 signalling but stimulates TGFβ-ALK5-Smad2/3 signalling in pulmonary ECs, which may lead to impaired pulmonary angiogenesis in developing lungs.