Endothelial-to-mesenchymal transition (EndMT) plays a critical role in the flow-induced vascular remodeling process, such as pulmonary arterial hypertension (PAH) related to congenital heart disease (CHD). NBL1 (neuroblastoma suppressor of tumorigenicity 1) is a secreted glycoprotein that has been implicated in CHD–PAH by aggravating the phenotypic transformation of smooth muscle cells. However, the underlying mechanisms regarding the interplay between NBL1 and endothelial cells in CHD–PAH remain to be fully elucidated. Thus, we aimed to identify the potential effect of NBL1 on EndMT using a novel flow-associated PAH model with Nbl1 knockout rats. The phenotype of EndMT was detected using RNA sequencing and further examined using western blotting and immunostaining of pulmonary arteries. Our observations demonstrated that the novel strategy of Nbl1 knockout effectively attenuated flow-associated PAH through downregulation of EndMT to some extent. Mechanistic experiments were established on human pulmonary artery endothelial cells to confirm that EndMT was induced by NBL1 in vitro. After 7 days’ stimulation with NBL1, concentrations of EndMT-related biomarkers and downstream transcription factors were quantified using RNA sequencing, western blotting, and immunocytochemistry. Both in vitro and in vivo experiments supported the imbalance of increased TGF-β (transforming growth factor-β) and dysregulation of BMP (bone morphogenetic protein) signaling by NBL1. Blocking the canonical TGF-β pathway efficiently preserved endothelial function upon NBL1 stimulation. These data suggested that NBL1 aggravated flow-associated PAH by inducing EndMT via the TGF-β and BMP signaling pathway. Thus, antagonizing NBL1 and rebalancing TGF-β and BMP signaling may be a suitable therapeutic target for CHD–PAH.