Abstract: Chromosome segregation is triggered by separase, an enzyme that cleaves cohesin, the protein complex that holds sister chromatids together. Separase activation requires the destruction of its inhibitor, securin, which occurs only upon the correct attachment of chromosomes to the spindle. However, other mechanisms restrict separase activity to the appropriate window in the cell cycle because cohesin is cleaved in a timely manner in securin-deficient cells. We investigated the mechanism by which the protector protein Shugoshin counteracts cohesin cleavage in budding yeast. We show that Shugoshin can prevent separase activation independently of securin. Instead, PP2A(Cdc55) is essential for Shugoshin-mediated inhibition of separase. Loss of both securin and Cdc55 leads to premature sister chromatid separation, resulting in aneuploidy. We propose that Cdc55 is a separase inhibitor that acts downstream from Shugoshin under conditions where sister chromatids are not under tension.