EphA and EphB receptors preferentially bind ephrin-A and ephrin-B ligands, respectively, but EphA4 is exceptional for its ability to bind all ephrins. Here, we report the crystal structure of the EphA4 ligand-binding domain in complex with ephrin-B2, which represents the first structure of an EphA-ephrin-B interclass complex. A loose fit of the ephrin-B2 G-H loop in the EphA4 ligand-binding channel is consistent with a relatively weak binding affinity. Additional surface contacts also exist between EphA4 residues Gln(12) and Glu(14) and ephrin-B2. Mutation of Gln(12) and Glu(14) does not cause significant structural changes in EphA4 or changes in its affinity for ephrin-A ligands. However, the EphA4 mutant has approximately 10-fold reduced affinity for ephrin-B ligands, indicating that the surface contacts are critical for interclass but not intraclass ephrin binding. Thus, EphA4 uses different strategies to bind ephrin-A or ephrin-B ligands and achieve binding promiscuity. NMR characterization also suggests that the contacts of Gln(12) and Glu(14) with ephrin-B2 induce dynamic changes throughout the whole EphA4 ligand-binding domain. Our findings shed light on the distinctive features that enable the remarkable ligand binding promiscuity of EphA4 and suggest that diverse strategies are needed to effectively disrupt different Eph-ephrin complexes.