Mouse protein-25 (MO25) proteins are crucial in development and morphogenesis from plants to humans. Fission yeast MO25 protein Pmo25 is essential for cell polarity and division. However, how Pmo25 regulates cytokinesis remains largely unknown. We identified and confirmed that Pmo25 binds to the Munc13/UNC-1 3 family protein Ync13 through yeast two-hybrid screen, co-immunoprecipitation, and in-vitro binding assays. In pmo25 mutants, contractile-ring stability and septation are defective during cytokinesis; the rings slide on the plasma membrane and some disintegrate before or during constriction. Moreover, Pmo25 interacts directly with the myosin-II light chain Cdc4, which is essential for the contractile-ring assembly and functions. Furthermore, division-site recruitment of the NDR kinase Sid2 in the Septation Initiation Network and the glucanase Eng1 for daughter-cell separation are compromised in pmo25 mutants, consistent with the cytokinesis defects. Our findings reveal a novel mechanism on how Pmo25 regulates cytokinesis and suggest that conserved MO25 proteins can link various cellular processes.