Before fertilization, ovulated mammalian oocytes are arrested at the metaphase of second meiosis (MIl), which is maintained by the so-called cytostatic factor (CSF). It is well known that the continuous synthesis and accumulation of cyclin B is critical for maintaining the CSF-mediated MIL arrest. Recent studies by us and others have shown that Ccnb3 is required for the metaphase-to-anaphase transition during the first meiosis of mouse oocytes, but whether Ccnb3 plays a role in MIL arrest and exit remains unknown. Here, we showed that the protein level of Ccnb3 gradually decreased during oocyte meiotic maturation, and exogenous expression of Ccnb3 led to release of MIL arrest, degradation of securin, separation of sister chromatids, extrusion of the second polar body (PB2), and finally entry into interphase. These phenotypes could be rescued by inhibition of Weel B or CDK2. Our results indicate that Ccnb3 plays a critical regulatory role in MIL arrest and exit in mouse oocytes. (C) 2019 Elsevier Inc. All rights reserved.