Separase, the cysteine protease that cleaves cohesin and thereby triggers chromosome disjunction, is inhibited by both securin- and phosphorylation-dependent cyclin B1 binding. Using a novel phosphorylation-specific antibody, we show that mitotic-specific phosphorylation of human separase on S1126 is required to establish, but not maintain, cyclin B1 binding. Cells expressing a non-phosphorylatable S1126A mutant maintain cohesion early in mitosis, aligning their chromosomes. Cohesion is then synchronously lost 5 minutes ahead of schedule, without degrading securin or cyclin B1. This premature chromatid disjunction requires the catalytic activity of separase, indicating that it is dependent on cohesin cleavage. Single chromatids then attempt to realign but the lack of tension results in unstable kinetochore-microtubule interactions and Aurora-B-dependent spindle checkpoint activation. Separase mutants that cannot bind cyclin B1 but are phosphorylated on S1126 phenocopy separase S1126A, indicating that cyclin B1 binding, rather than phosphorylation, is the key inhibitory event. Significantly, by overexpressing separase S1126A, we have simultaneously overridden the two known inhibitory mechanisms. First, by elevating separase levels above securin, securin-mediated inhibition is alleviated. Second, by preventing phosphorylation, cyclin-B1-mediated inhibition is also alleviated. Surprisingly, however, cohesion is maintained during the early stages of mitosis, indicating the existence of another mechanism that either inhibits separase or protects its substrate during early mitosis.