Targeting the tumor vasculature with antibody-drug conjugates (ADCs) is a promising anti-cancer strategy that in order to be realized must overcome several obstacles, including identification of suitable targets and optimal warheads. Here, we demonstrate that the cell-surface protein CD276/B7-H3 is broadly overexpressed by multiple tumor types on both cancer cells and tumor-infiltrating blood vessels, making it a potentially ideal dual-compartment therapeutic target. In preclinical studies CD276 ADCs armed with a conventional MMAE warhead destroyed CD276-positive cancer cells, but were ineffective against tumor vasculature. In contrast, pyrrolobenzodiazepine-conjugated CD276 ADCs killed both cancer cells and tumor vasculature, eradicating large established tumors and metastases, and improving long-term overall survival. CD276-targeted dual-compartment ablation could aid in the development of highly selective broad-acting anti-cancer therapies. Display omitted •CD276/B7-H3 is broadly overexpressed in both cancer cells and tumor vasculature•Both angiogenic and non-angiogenic tumor vasculature express CD276•Anti-CD276-drug conjugates display potent anti-tumor and anti-metastatic activity•Pyrrolobenzodiazepine dimers are optimal warheads for targeting tumor vasculature Seaman et al. show that CD276 is broadly overexpressed in cancer cells and tumor vascular cells and demonstrate anti-CD276-drug conjugates as promising anti-cancer reagents. The drug selected for conjugation is important because tumor vascular cells can be resistant to a drug to which tumor cells are sensitive.