Chromosomal segregation and cytokinesis are tightly regulated processes required for successful cell division. The cysteine protease separase cleaves a subunit of the cohesin complex to allow chromosome segregation at anaphase onset. Separase also regulates meiotic cortical granule exocytosis and vesicle trafficking during cytokinesis, both of which involve RAB-11. Separase has non-proteolytic signaling functions in addition to its role in substrate cleavage, and its mechanism in exocytosis is unknown. We sought to determine whether separase regulates RAB-11 vesicle exocytosis through a proteolytic or non-proteolytic mechanism. To address this question, we generated a protease-dead separase, SEP-1PD::GFP, and unexpectedly found that it is dominant negative. Consistent with its role in cohesin cleavage, SEP-1PD::GFP causes chromosome segregation defects. Depletion of the substrate subunit of cohesin rescues this defect, suggesting that SEP-1PD::GFP impairs cohesin cleavage by a substrate trapping mechanism. We investigated whether SEP-1PD::GFP also impairs RAB-11 vesicle trafficking. SEP-1PD::GFP causes a low rate of cytokinesis failure that is synergistically exacerbated by depletion of the core exocytic t-SNARE protein SYX-4. Interestingly, SEP-1PD::GFP causes an accumulation of RAB-11 vesicles at the cleavage furrow site and delayed the exocytosis of cortical granules during anaphase I. Depletion of SYX-4 further enhanced RAB-11::mCherry and SEP-1PD::GFP plasma membrane accumulation during cytokinesis. These findings suggest that the protease activity of separase is required for the exocytosis of RAB-11 vesicles during cortical granule exocytosis and mitotic cytokinesis.