ABSTRACT Centrosome cohesion, mostly regarded as a proteinaceous linker between parental centrioles, ensures that the interphase centrosome(s) function as a single microtubule-organizing center. Impairment of centrosome cohesion leads to the splitting of centrosomes. Although the list of cohesion proteins is growing, the precise composition and regulation of centrosome cohesion are still largely unknown. In this study, we show that the centriolar protein centlein (also known as CNTLN) localizes to the proximal ends of the centrioles and directly interacts with both C-Nap1 (also known as Cep250) and Cep68. Moreover, centlein complexes with C-Nap1 and Cep68 at the proximal ends of centrioles during interphase and functions as a molecular link between C-Nap1 and Cep68. Depletion of centlein impairs recruitment of Cep68 to the centrosomes and, in turn, results in centrosome splitting. Both centlein and Cep68 are novel Nek2A substrates. Collectively, our data demonstrate that centrosome cohesion is maintained by the newly identified complex of C-Nap1–centlein–Cep68.