Oncogenic fusions are major drivers in leukemogenesis and may serve as potent targets for treatment. DUX4/IGHs have been shown to trigger the abnormal expression of ERGalt through binding to DUX4-Responsive-Element (DRE), which leads to B-cell differentiation arrest and a full-fledged B-ALL. Here, we determined the crystal structures of Apo- and DNADRE-bound DUX4HD2 and revealed a clamp-like transactivation mechanism via the double homeobox domain. Biophysical characterization showed that mutations in the interacting interfaces significantly impaired the DNA binding affinity of DUX4 homeobox. These mutations, when introduced into DUX4/IGH, abrogated its transactivation activity in Reh cells. More importantly, the structure-based mutants significantly impaired the inhibitory effects of DUX4/IGH upon B-cell differentiation in mouse progenitor cells. All these results help to define a key DUX4/IGH-DRE recognition/step in B-ALL.