AbstractThe dynamic polymerization and depolymerization of actin filaments is essential for various cellular processes such as cell migration, rotation, cytokinesis, and mammalian oocyte maturation. Tropomodulin 3 (Tmod3) binds to the slow-growing (pointed) ends of the actin filament, thereby protecting the filament from depolymerization. However, the roles of Tmod3 in mammalian oocyte maturation remain elusive. Tmod3 mRNA and protein is present at all stages of mouse oocyte maturation. Tmod3 protein is mainly localized in the cytoplasm and appears enriched near the chromosome during maturation. By knocking down or ectopically overexpressing Tmod3, we confirmed that Tmod3 regulate the level of the intracytoplasmic actin mesh and asymmetric spindle migration. Expression of N-terminal Tmod3 (correspond to 1–155 amino acids), which contains the tropomyosin-binding site, results in decreased density of the actin mesh, thereby demonstrating the importance of the interaction between tropomyosin and tropomodulin for the maintenance of the actin mesh. Taken together, these findings indicate that Tmod3 plays crucial roles in oocyte maturation, presumably by protecting the actin filament from depolymerization and thereby controlling the density of the cytoplasmic actin mesh.