Endothelin B receptor (ETBR) is a G protein-coupled receptor able to bind equally to the three identified human endothelin peptides. It is expressed primarily on vascular endothelial cells and involved in various physiological processes including vascular tone homeostasis, enteric nervous system development, melanogenesis and angiogenesis. Furthermore, overactivation or overexpression of ETBR have been associated with the development of various diseases such as cardiovascular disorders and cancers. Therefore, ETBR appears to be relevant target for the therapy or diagnosis of highly prevalent human diseases. In this study, we report the in vitro characterization of rendomab-B1, a monoclonal antibody (mAb) obtained by genetic immunization, which selectively recognizes the native form of human ETBR (hETBR). Rendomab-B1 is the first-reported mAb that behaves as a potent antagonist of hETBR. It recognizes an original extracellular conformational epitope on the receptor, distinct from the endothelin-1 (ET-1) binding site. Rendomab-B1 not only blocks ET-1-induced calcium signaling pathway and triggers rapid receptor internalization on recombinant hETBR-expressing cells, but also exerts pharmacological activities on human vascular endothelial cells, reducing both cell viability and ET-1-induced hETBR synthesis. In addition, binding experiments using rendomab-B1 on different melanoma cell lines reveal the structural and functional heterogeneity of hETBR expressed at the surface of these cancer cells, strongly suggesting the existence of tumor-specific receptors. Collectively, our results underscore the value of rendomab-B1 for research, therapeutic and diagnostic applications dealing with hETBR.