PURPOSE Endoglin (ENG; CD105) is a co-receptor of the transforming growth factor-$\beta$ (TGF-$\beta$) family that is highly expressed in proliferating endothelial cells. Often coopted by cancer cells, ENG can lead to neo-angiogenesis and vasculogenic mimicry in aggressive malignancies. It exists both as a transmembrane cell surface protein, where it primarily interacts with TGF-$\beta$, and as a soluble matricellular protein (sENG) when cleaved by matrix metalloproteinase 14 (MMP14). High ENG expression has been associated with poor prognosis in Ewing sarcoma (ES), an aggressive bone cancer that primarily occurs in adolescents and young adults. However, the therapeutic value of ENG-targeting has not been fully explored in this disease. EXPERIMENTAL DESIGN We characterized the expression pattern of transmembrane ENG, sENG and MMP14 in preclinical and clinical samples. Subsequently, the antineoplastic potential of two novel ENG-targeting monoclonal antibody-drug conjugates (ADCs), OMTX503 and OMTX703, which differed only by their drug payload (nigrin-b A chain and cytolysin, respectively), was assessed in cell lines and preclinical animal models of ES. RESULTS Both ADCs suppressed cell proliferation in proportion to the endogenous levels of ENG observed in vitro Moreover, the ADCs significantly delayed tumor growth in ES cell line-derived xenografts and patient-derived xenografts (PDXs) in a dose-dependent manner. CONCLUSIONS Taken together, these studies demonstrate potent preclinical activity of first-in-class anti-ENG ADCs as a nascent strategy to eradicate ES.