Nek2 is a mitotic kinase with multiple cellular functions involving phosphorylation of diverse substrates. Suppression of Nek2 in early mouse embryos has been shown to arrest development at the 4-cell stage with defects in mitotic spindle assembly as well as in interphase nuclear morphology. In the present study, we suppressed expression of two Nek2 centrosomal substrates, Nip2 and C-Nap1, in early mouse embryos. The development of the Nip2-suppressed embryo was arrested at the 4-cell stage with mitotic defects in the blastomeres. In contrast, C-Nap1 suppression did not produce a visible phenotype. The phenotypic similarities of the Nip2- and Nek2-suppressed embryos suggest that Nip2 may be a substrate of Nek2 that is required for mitotic spindle assembly in early mouse embryos.