Remodeling of the embryo surface after fertilization is mediated by the exocytosis of cortical granules derived from the Golgi complex. This process is essential for oocyte-to-embryo transition in many species. However, how the fertilization signal reaches the cortical granules for their timely exocytosis is largely unknown. In Caenorhabditis elegans, the recruitment of separase, a downstream effector of the fertilization signal, to the cortical granules is essential for exocytosis because separase is required for membrane fusion. However, the molecule that recruits separase to the cortical granules remains unidentified. In this study, we found that Rab6, a Golgi-associated GTPase, is essential to recruit separase to the cortical granules in C. elegans embryos. Knockdown of the rab-6.1 gene, a Rab6 homologue in C. elegans, resulted in failure of the membrane fusion step of cortical granule exocytosis. Using a transgenic strain that expresses GFP-fused RAB-6.1, we found that RAB-6.1 temporarily co-localized with separase on the cortical granules for a few minutes and then was dispersed in the cytoplasm concomitantly with membrane fusion. We found that RAB-6.1 as well as cyclin-dependent kinase (CDK)-1 and anaphase promoting complex/cyclosome (APC/C) was required to recruit separase to the cortical granules. RAB-6.1 was not required for the chromosome segregation process, unlike CDK-1, APC/C, and SEP-1. The results indicate that RAB-6.1 is required specifically for the membrane fusion step of exocytosis and for the recruitment of separase to the granules. Thus, RAB-6.1 is an important molecule for the timely exocytosis of the cortical granules during oocyte-to-embryo transition.