The Bre5 protein is a cofactor for the deubiquitynating enzyme Ubp3 and it contains an NTF2 like protein recognition module that is essential for Ubp3 activity. In this study, we report the X-ray crystal structure of the Bre5 NTF2-like domain and show that it forms a homodimeric structure that is similar to other NTF2-like domains, except for the presence of an interatomic disulfide bond in the crystals. Sedimentation equilibrium studies reveals that under non reducing conditions the Bre5 NTF2-like domain is exclusively dimeric while a disulfide bond deficient mutant undergoes a monomer-dimer equilibrium with a dissociation constant in the mid nanomolar range, suggesting that dimer formation, and possibly also disulfide bond formation, may modulate Bre5 function in vivo. Using deletion analysis, we also identify a novel N-terminal domain of Ubp3 that is necessary and sufficient for interaction with Bre5 and use isothermal titration calorimetry to show that Bre5 and Ubp3 form a 2:1 complex, in contrast to other reported NTF2-like domain/protein interactions that form 1:1 complexes. Finally, we employ structure-based mutagenesis to map the Ubp3 binding surface of Bre5 to a region near the Bre5 dimer interface and show that this binding surface of Bre5 is important for Ubp3 function in vivo. Together, these studies provide novel insights into protein recognition by NTF2-like domains and provide a molecular scaffold for understanding how Ubp3 function is regulated by Bre5 cofactor binding.