Endoglin (ENG) promotes angiogenesis by enhancing activation of TGF-beta type I receptors ALK-1 and ALK-5. ALK-1 phosphorylates transcription factors SMAD1/5, which bind to BMP-responsive elements (BRE), whereas ALK-5 phosphorylates SMAD3, which binds to CAGA elements. Expression of ENG is increased during myocardial infarction (MI). We investigated which ENG signaling pathway is activated in endothelial cells during hypoxia. Expression of ENG, ALK-1, ALK-5, and phosphorylated SMAD1/3/5 by immunostaining and immunoblotting in a mouse model of myocardial infarction (MI) and in hypoxic human aortic endothelial cells (HAECs) was evaluated. Activation of BRE and CAGA was measured by luciferase assays in cells transfected with plasmids expressing ENG or ALK-1 and the number of cells was quantified. mRNA expression of the target genes of TGF-beta signaling, ID1 and BCL-X, was quantified by real-time RT-PCR. Expression of ENG, ALK-1 and phosphorylated SMAD1/5, but not ALK-5 or phosphorylated SMAD3, was significantly increased in hypoxic endothelial cells in vivo and in vitro. Overexpression of both ENG and ALK-1 significantly increased BRE but not CAGA activity, expression of ID1 and BCL-X and the number of HAECs at hypoxia. ENG/ALK-1 signaling is one of the factors that regulate endothelial cell activity during adaptive cardiac angiogenesis.