Never in mitosis A-related kinase 2A (Nek2A), a centrosomal serine/threonine kinase, is involved in mitotic progression by regulating the centrosome cycle. Particularly, Nek2A is necessary for dissolution of the intercentriole linkage between the duplicated centrosomes prior to mitosis. Nek2A activity roughly parallels its cell cycle-dependent expression levels, but the precise mechanism regulating its activity remains unclear. In this study, we found that γ-taxilin co-localized with Nek2A at the centrosome during interphase and interacted with Nek2A in yeast two-hybrid and pull-down assays and that γ-taxilin regulated centrosome disjunction in a Nek2A-dependent manner. γ-Taxilin depletion increased the number of cells with striking splitting of centrosomes. The precocious splitting of centrosomes induced by γ-taxilin depletion was attenuated by Nek2A depletion, suggesting that γ-taxilin depletion induces the Nek2A-mediated dissolution of the intercentriole linkage between the duplicated centrosomes nevertheless mitosis does not yet begin. Taken together with the result that γ-taxilin protein expression levels were decreased at the onset of mitosis, we propose that γ-taxilin participates in Nek2A-mediated centrosome disjunction as a negative regulator through its interaction with Nek2A.