Updated project metadata. Eukaryotic ribosome synthesis involves more than 200 assembly factors, which promote ribosomal RNA (rRNA) processing, modification and folding, and assembly of ribosomal proteins. The formation and maturation of the earliest pre-60S particles requires structural remodeling by the Npa1 complex, but is otherwise still poorly understood. Here we introduce Rbp95, a constituent of early pre-60S particles, as a novel ribosome assembly factor that likely already binds before the 90S to pre-60S transition. We show that Rbp95 is both genetically and physically linked to most Npa1 complex members, and to ribosomal protein Rpl3. Notably, the RNA-binding protein Rbp95 associates with helix H95 in the 3’ region of the 25S rRNA, in close proximity to the binding sites of Npa1 and Rpl3. Additionally, Rbp95 interacts with several snoRNAs. In the absence of Rbp95, proteins binding in proximity to helix H95 are less efficiently recruited to early pre-60S particles. Moreover, combined mutation of Rbp95 and Npa1 complex members leads to a delay in the formation of early pre-60S particles. We propose that Rbp95 acts together with the Npa1 complex in the 90S to pre-60S transition and in promoting pre-rRNA folding events within pre-60S particles that facilitate the recruitment of subsequent assembly factors.