In eukaryotic cells, replicated DNA strands remain physically connected until their segregation to opposite poles of the cell during anaphase. This "sister chromatid cohesion" is essential for the alignment of chromosomes on the mitotic spindle during metaphase. Cohesion depends on a multisubunit protein complex called cohesin, which possibly forms the physical bridges that connect sisters. Proteolytic cleavage of cohesin's Scc1 subunit at the metaphase to anaphase transition is essential for sister chromatid separation and depends on a conserved protein called separin. We show here that separin is a cysteine protease related to caspases and that it alone can cleave Scc1 in vitro. By replacing one of Scc1's cleavage sites by that for a different site specific protease, we show that cleavage of Scc1 in metaphase arrested cells is sufficient to trigger the separation of sister chromatids and their segregation to opposite cell poles.