ACE2 and Ang-1–7 have been shown to protect against pulmonary hypertension (PH). Mechanisms for this remain unclear. Considering the important role of ET-1 in the pathophysiology of PH and endothelial dysfunction, we questioned whether Ang-(1–7) influences ET-1 signaling in endothelial cells and whether Ang-(1–7) treatment influences the ET-1 system in PH. Human microvascular endothelial cells (HMEC) were stimulated with ET-1 in the absence/presence of Ang 1–7 and showed that Ang 1–7 increased preproET-1 mRNA levels, ET-1 release, and ETBR protein levels. ET-1 increases in e-selectin mRNA, VCAM-1 protein and TNFα production were blocked by Ang 1–7. Pro-inflammatory effects were dependent on NO production. Ang 1–7 increased NO production in a Mas and ETBR-dependent manner. An interaction between Mas and ETBR was observed by immunoprecipitation. To further characterise a physical interaction between Mas/ETBR, we utilised novel technology, employing a library of overlapping peptides scanning the entirety of the MasR sequence, to define the interaction sites for ETBR binding. By substitution or sequence truncation we identified two distinct regions on the MasR that confer specificity for ETBR binding. Peptides that disrupt each of these regions to prevent Mas/ETBR interaction were developed for in vitro validation. To investigate the pathophysiological significance of our findings, we investigated whether Ang-(1–7) treatment ameliorates PH and whether this is associated with changes in ET-1 status. Hypobaric hypoxia was used to induce PH in mice, which were divided in 4 groups: normoxic controls (NC), hypoxic PH (HP), normoxic (NA) and hypoxic PH (HA) treated with orally active Ang 1–7 30 μg/kg/day for 14 days. In HP mice, RVSP, RVH and ET-1 levels were increased and blocked by Ang 1–7 treatment. Hyper-contractility and endothelial dysfunction in pulmonary arteries of HP mice compared to NC was attenuated by Ang 1–7. These findings indicate that vasoprotective effects of Ang-(1–7) may be mediated through dimerization of MAS:ETBR. In vivo studies support a relationship between the Ang-(1–7)/MAS and ET-1 systems. In conclusion we have identified a novel link between Ang-(1–7) and ET-1 through physical interactions between MAS and ETBR.