During the early steps of ribosome biogenesis in mammals, the two ribosomal subunits 40S and 60S are produced via splitting of the large 90S pre-ribosomal particle (90S) into pre-40S and pre-60S pre-ribosomal particles (pre-40S and pre-60S). We previously proposed that replacement of fibrillarin by Nop52 (RRP1/NNP-1) for the binding to p32 (C1QBP) is a key event that drives this splitting process. However, how the replacement by RRP1 is coupled with the endo- and/or exo-ribonucleolytic cleavage of pre-rRNA remains unknown. In this study, we demonstrate that RRP1 deficiency suppressed site 2 cleavage on ITS1 of 47S/45S, 41S and 36S pre-rRNAs in human cells. RRP1 was also present in 90S and was localized in the dense fibrillar component of the nucleolus dependently on active RNA polymerase I transcription. In addition, double knockdown of XRN2 and RRP1 revealed that RRP1 accelerated the site 2 cleavage of 47S, 45S and 41S pre-rRNAs. These data suggest that RRP1 is involved not only in competitive binding with fibrillarin to C1QBP on 90S but also in site 2 cleavage in ITS1 of pre-rRNAs at early stages of human ribosome biogenesis; thus, it is likely that RRP1 integrates the cleavage of site 2 with the physical split of 90S into pre-40S and pre-60S.