In brief Mammalian zygotes often fail to unify their genomes. This study showed that the position of the centrosome is crucial for regulation of pronuclear apposition and genome unification. Abstract After the sperm enters the ovum, male and female pronuclei carrying the genetic information of both parents are formed. These pronuclei migrate to the center of the ovum, followed by genome unification upon mitosis onset. However, the genomes of some gametes fail to unify. The mechanism by which parental genetic information fails to unify is not yet fully understood. Here, we used immunofluorescence staining and live imaging techniques to study pronuclear migration in fertilized bovine ova. Microtubules play a more important role in pronuclear migration than actin. Centrosomal microtubules regulate the localization of motor dynein bound to the pronuclear membrane. The presence of at least one centrosome between the male and female pronuclei is necessary for apposition of the two pronuclei and genome unification. Our data highlight the importance of centrosome positioning in the apposition of pronuclei and parental genome unification.