Eph receptor B2 (EphB2) overexpression is associated with poor clinical outcomes in various tumors. EphB2 is involved in malignant tumor progression through the promotion of invasiveness and metastasis. Genetic and transcriptome analyses implicated that EphB2 is a therapeutic target for specific tumor types. A monoclonal antibody (mAb) is one of the essential therapeutic strategies for EphB2-positive tumors. We previously developed an anti-EphB2 mAb, EbMab-12 (IgG, kappa), by immunizing mice with EphB2-overexpressed glioblastoma. EbMab-12 specifically reacted with the EphB2-overexpressed Chinese hamster ovary-K1 (CHO/EphB2) and some cancer cell lines in flow cytometry. In this study, we engineered EbMab-12 into a mouse IgG type (EbMab-12-mG) and a human IgG-type (EbMab-12-hG) mAb. EbMab-12-mG and EbMab-12-hG retained the reactivity to EphB2-positive cells and exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in the presence of effector cells and complements, respectively. In CHO/EphB2, triple-negative breast cancer, and lung mesothelioma xenograft models, both EbMab-12-mG and EbMab-12-hG exhibited potent antitumor efficacy. These results indicated that EbMab-12-derived mAbs could be applied to mAb-based therapy against EphB2-positive tumors.