14-3-3ε and 14-3-3γ localize to the centrosome and regulate centrosome duplication, by inhibiting cdc25C function. As 14-3-3γ and 14-3-3ε form a complex with centrosomal proteins, we asked if this ability was required to regulate centrosome duplication. The results in this report demonstrate that 14-3-3ε and 14-3-3γ form a complex with Centrin2 and that the binding site is located in the N-terminal EF hand in Centrin2, EF1. A Centrin2 mutant that does not form a complex with 14-3-3 proteins displays a punctate cytoplasmic localization and does not localize to the centrosome. These results suggest that in addition to negatively regulating centrosome duplication as previously reported, 14-3-3 proteins might also be required for centriole biogenesis by regulating the localization of Centrin2 at the centrosome.