Background：Bronchopulmonary dysplasia (BPD) is a chronic lung disease of premature infants that involves pulmonary vascular development disorder as the main pathological feature; hyperoxia is its main etiology. Twist1 strictly controls the development of blood vessels via the Tie2-Angs signaling axis. However, previous research on Twist1 mainly focuses on various tumors; its effect on BPD has yet to be reported. The present study represents the first investigation of the role and related mechanisms of the Twist1-Tie2-Angs signaling pathway in hyperoxia-induced endothelial cell injury. Methods: Primary human umbilical vein endothelial cells were used as an in vitro model. A Twist1 inhibitor (harmine) was applied to normal and hyperoxia-exposed endothelial cells. Then, we observed the permeability and tubule formation ability of endothelial cells after reducing Twist1 protein. Results: Hyperoxia increased the permeability of endothelial cells and decreased tubule formation ability. Under physiological conditions dominated by angiogenin 1 (Ang1), reducing the expression of Twist1 increases the permeability of endothelial cells and reduces tubule formation ability. In contrast, under hyperoxia conditions dominated by angiogenin 2 (Ang2), reducing the expression of Twist1 reduced the permeability of endothelial cells and increased tubule formation ability. Conclusion: Twist1 depends on the balance of Ang1 and Ang2 to control the permeability and tubule formation of endothelial cells. Reducing the levels of Twist1 may be a protective mechanism for BPD.