<p>BubR1 is a key component of the mitotic checkpoint, a surveillance mechanism which ensures accurate chromosome segregation by facilitating the assembly of the mitotic checkpoint complex (MCC) and promoting its binding to the anaphase-promoting complex/cyclosome (APC/C). Although BubR1’s role in the mitotic checkpoint has been extensively investigated, the function of its N-terminal tetratricopeptide repeat (TPR) domain remains poorly understood. In this study, we first establish the essential role of the BubR1 TPR domain in the mitotic checkpoint. Guided by the resolved cryo-EM structure of the MCC-APC/C complex, we identify and characterize several interactions between the BubR1 TPR domain and Mad2, Cdc20^APC/C and Apc2. Furthermore, we discover an intramolecular interaction between the TPR domain and downstream residues of BubR1, which appears to organize a structure resembling a “lasso” that incorporates four Cdc20^APC/C-binding elements, thereby enhancing engagement with Cdc20 in the APC/C. Functional and biochemical analyses demonstrate that these interactions collectively promote MCC assembly and MCC-APC/C binding, enabling rapid SAC activation in response to microtubule-kinetochore attachment defects.</p>