The AAA-ATPase thyroid hormone receptor interacting protein 13 (TRIP13), jointly with the Mad2-binding protein p31^comet, promotes the inactivation of the mitotic (spindle assembly) checkpoint by disassembling the mitotic checkpoint complex (MCC). This checkpoint system ensures the accuracy of chromosome segregation by delaying anaphase until correct bipolar attachment of chromatids to the mitotic spindle is achieved. MCC inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets for degradation securin, an inhibitor of anaphase initiation. MCC is composed of the checkpoint proteins Mad2, BubR1, and Bub3, in association with the APC/C activator Cdc20. The assembly of MCC in active checkpoint is initiated by the conversion of Mad2 from an open (O-Mad2) to a closed (C-Mad2) conformation, which then binds tightly to Cdc20. Conversely, the disassembly of MCC that takes place when the checkpoint is turned off involves the conversion of C-Mad2 back to O-Mad2. Previously, we found that the latter process is mediated by TRIP13 together with p31^comet, but the mode of their interaction remained unknown. Here, we report that the oligomeric form of TRIP13 binds both p31^comet and MCC. Furthermore, p31^comet and checkpoint complexes mutually promote the binding of each other to oligomeric TRIP13. We propose that p31^comet bound to C-Mad2–containing checkpoint complex is the substrate for the ATPase and that the substrate-binding site of TRIP13 is composed of subsites specific for p31^comet and C-Mad2–containing complex. The simultaneous occupancy of both subsites is required for high-affinity binding to TRIP13.