Ephrin–EPH signalling, which requires cell–cell interaction as ephrin ligands and EPH receptors are both membrane bound, regulates tissue separation in embryogenesis. Hwang et al. show that ephrin B1 interacts with the E3 ligases SMAD ubiquitylation regulatory factor 1 (SMURF1) and SMURF2, and that SMURF2 promotes ephrin B1 degradation. Ephrin B1 was stable when SMURF1 and SMURF2 were co-expressed in Xenopus laevis embryos, suggesting that SMURF1 inhibits SMURF2-mediated ephrin B1 degradation. To assess ephrin B1-dependent separation of mesoderm from ectoderm, the authors introduced morpholinos against SMURF1 or ephrin B1 into mesoderm and found that tissue separation was inhibited. The co-introduction of morpholinos against SMURF1 and SMURF2 stabilized ephrin B1 and rescued this phenotype. Thus, SMURF2 regulates ephrin B1–EPH signalling during development by promoting ephrin B1 degradation; SMURF1 antagonizes SMURF2 in this setting.