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The spindle assembly checkpoint (SAC) restrains anaphase until all chromosomes become bi‐oriented on the mitotic spindle. The SAC protein Mad2 can fold into two distinct conformers, open (O) and closed (C), and can asymmetrically dimerize. Here, we describe a monoclonal antibody that specifically recognizes the dimerization interface of C‐Mad2. This antibody revealed several conformation‐specific features of Mad2 in human cells. Notably, we show that Mad2 requires association with Mad1 to adopt the closed conformation and that the activity of the Mad1:C‐Mad2 complex undergoes regulation by p31comet‐dependent ‘capping’. Furthermore, C‐Mad2 antibody microinjection caused an abrupt termination of the SAC and accelerated mitotic progression. Remarkably, microinjection of a Mad1‐neutralizing antibody triggered a comparable mitotic acceleration. Our study provides direct in vivo evidence for the model that a kinetochore complex of Mad1:C‐Mad2 acts as a template to sustain the SAC and it challenges the distinction between SAC and mitotic timer.