Centriole disengagement is considered an essential step for licensing a new round of centriole duplication in the next cell cycle. Separase is critical for centriole disengagement. Here, we showed that pericentrin B (PCNTB) is specifically cleaved by separase at the exit of mitosis. The cleavage-resistant PCNTB mutant blocks the centriole disengagement and duplication. We also observed that an artificial cleavage of PCNTB during M phase induced premature disengagement of centrioles. Based on these results, we concluded that the separase-dependent cleavage of PCNTB is necessary and sufficient for centriole disengagement during mitosis.