In Xenopus somatic cell nuclear cloning, the nucleoli of donor nuclei rapidly and almost completely disappear in egg cytoplasm. We previously showed that the germ cell-specific proteins FRGY2a and FRGY2b were responsible for this unusually drastic nucleolar disassembly. The nucleolar disassembly occurs without inhibition of pre-rRNA transcription, a well known trigger for nucleolar segregation, and the mechanism for the nucleolar disassembly by FRGY2a and FRGY2b remains largely unknown. In this study, we searched for FRGY2a-interacting proteins and investigated the functional consequences of their interactions through a series of experiments. We showed that during the nucleolar disassembly, FRGY2a localized to the nucleoli of isolated nuclei and was capable of disassembling purified nucleoli, suggesting a direct interaction between FRGY2a and nucleolar components. Using a His tag pulldown approach, we identified the abundant and multifunctional nucleolar protein B23 as a potential target of FRGY2a and its related human protein YB1. A specific interaction between FRGY2a/YB1 and B23 was confirmed by co-immunoprecipitation. Finally, B23 knockdown using short interfering RNA and a subsequent add-back experiment confirmed that B23 was necessary for nucleolar disassembly by YB1. We propose that FRGY2a and YB1 disassemble nucleoli by sequestering B23, which is associated with pre-ribosomes and other structurally important nucleolar components.