Abstract: Spo13 is a key meiosis-specific regulator required for centromere cohesion and co-orientation, and for progression through two nuclear divisions. We previously reported that it causes a G2/M arrest, and may delay the transition from late anaphase to G1, when overexpressed in mitosis. Yet its mechanism of action has remained elusive. Here we show that Spo13, which is phosphorylated and stabilized at G2/M in a Cdk/Clb-dependent manner, acts at two stages during mitotic cell division. Its G2/M arrest is reversible and largely independent of the Mad2 spindle checkpoint. Genome-wide profiling suggests that its anaphase delay results from inhibition of Cdc14 function, since mRNAs whose induction requires Cdc14 activation are reduced. We show that the Spo13-induced delay in anaphase correlates with Cdc14 phosphatase retention in the nucleolus and with cyclin B accumulation, which both impede anaphase exit. At the onset of arrest, Spo13 is primarily associated with the nucleolus, where Cdc14 also accumulates. Significantly, overexpression of Separase (Esp1), which promotes G2/M and anaphase progression, suppresses Spo13 effects in mitosis, arguing that Spo13 acts upstream or parallel to Esp1. Combined with reduced Pds1 and cyclin B degradation, our findings are consistent with a role for Spo13 in regulating APC function, which controls both G2/M and anaphase. Similar effects of Spo13 during meiotic MI may prevent cell cycle exit and initiation of DNA replication prior to MII, thus ensuring two successive chromosome segregation events without an intervening S phase.