USP37 is a deubiquitinase (DUB) with roles in the regulation of DNA damage repair and the cohesion of sister chromatids during mitosis. USP37 contains a unique insert of three ubiquitin interacting motifs (UIMs) within its catalytic DUB domain. We investigated the role of the three UIMs in the ability of USP37 to cleave di-ubiquitin chains. We found that the third UIM of USP37 recognizes the proximal ubiquitin moiety of K48 di-Ub to potentiate cleavage activity and posit that this mechanism of action may be generalizable to other chain types. In the case of K48-linked ubiquitin chains this potentiation stemmed largely from a dramatic increase in catalytic rate (kcat). We also developed and characterized three ubiquitin variant (UbV) inhibitors that selectively engage distinct binding sites in USP37. In addition to validating the deduced functional roles of the three UIMs in catalysis, the UbVs highlight a novel and effective means to selectively inhibit members of the difficult to drug DUB family.