The receptor tyrosine kinase EphB4 is involved in tumor angiogenesis, proliferation, and metastasis. Designed ankyrin repeat proteins (DARPins) binding to the EphB4 extracellular domain were identified from a combinatorial library using phage display. Surface plasmon resonance (SPR) allowed us to distinguish between DARPins that either compete with the EphB4 ligand ephrin-B2 for binding to a common site or target a different epitope. The identified DARPins all prevent ligand-induced EphB4 phosphorylation and impair tube formation by endothelial cells in vitro. The competitive DARPin AB1 was additionally shown to inhibit vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-induced angiogenesis in vivo. In summary, we have isolated DARPins that exert antiangiogenic effects by specifically binding to EphB4 and may potentially lead to new cancer therapeutics.