Polo-like kinases (Plks) are a family of serine/threonine protein kinases that regulate multiple stages of mitosis. Expression and distribution of polo-like kinase 1 (Plk1) were characterized during porcine oocyte maturation, fertilization and early embryo development in vitro, as well as after microtubule polymerization modulation. The quantity of Plk1 protein remained stable during meiotic maturation. Plk1 accumulated in the germinal vesicles (GV) in GV stage oocytes. After germinal vesicle breakdown (GVBD), Plk1 was localized to the spindle poles at metaphase I (MI) stage, and then translocated to the middle region of the spindle at anaphase-telophase I. Plk1 was also localized in MII spindle poles and on the spindle fibers and on the middle region of anaphase-telophase II spindles. Plk1 was not found in the spindle region when colchicine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. After fertilization, Plk1 concentrated around the female and male pronuclei. During early embryo development, Plk1 was found to be in association with the mitotic spindle at metaphase, but distributed diffusely in the cytoplasm at interphase. Our results suggest that Plk1 is a pivotal regulator of microtubule organization and cytokinesis during porcine oocyte meiotic maturation, fertilization, and early embryo cleavage in pig oocytes.