Chromosomal segregation and cytokinesis are tightly regulated processes involved in cell division. The cysteine protease separase is required for proper chromosomal segregation. Separase cleaves a subunit of the protein complex called cohesin, which keeps the sister chromatids together until the end of metaphase. In addition, separase is a key player in meiotic cortical granule exocytosis and vesicle trafficking during mitotic cytokinesis. RAB-11 is known to regulate both cortical granule exocytosis and exocytosis during cytokinesis. RNAi knockdown of separase impairs RAB-11 vesicular trafficking during both events. This raises the question of whether or not separase’s protease activity regulates RAB-11 vesicle exocytosis. To address this question, we generated a catalytically inactive separase, SEP-1PD::GFP. We found that the expression of SEP-1PD::GFP causes chromosomal nondisjunction. Depletion of cohesin rescues this defect, indicating that cohesin cleavage is prevented by the inactive protease, possibly by a substrate trapping mechanism. To test whether SEP-1PD::GFP also impairs RAB-11 vesicular trafficking during cytokinesis, we imaged the embryos that express RAB-11::mCherry and SEP-1PD::GFP. Interestingly, expression of SEP-1PD::GFP causes an abnormal accumulation of RAB-11 vesicles at the cleavage furrow site, similar to depletion of separase by RNAi. Moreover, we found that RAB-11 co-localized with both wild type separase and our protease dead mutant on cortical granules during meiotic anaphase I. We filmed SEP-1PD::GFP expressing embryos during meiosis I to observe cortical granule exocytosis. Our results indicated that SEP-1PD::GFP expression delayed the completion of cortical granule exocytosis. We tested possible genetic interactions between SEP-1PD::GFP and core exocytic proteins. Our results indicate that the depletion of the t-SNARE syntaxin-4 (syx-4) further enhanced RAB-11::mCherry and SEP-1PD::GFP accumulation on the cleavage furrow and the midbody. Moreover, we found an additive increase in embryonic cytokinesis failure relative to syx-4 depletion or SEP-1PD::GFP expression alone. These findings suggest that the protease activity of separase is required for the exocytosis of RAB-11 vesicles during mitotic cytokinesis and cortical granule exocytosis during meiosis I.