The U3 box C/D snoRNA is one key element of 90S pre-ribosome. It contains a 5' domain pairing with pre-rRNA and the U3(B/C) and U3(C'/D) motifs for U3 packaging into a unique small nucleolar ribonucleoprotein particle (snoRNP). The RNA-binding protein Snu13/SNU13 nucleates on U3(B/C) the assembly of box C/D proteins Nop1p/FBL and Nop56p/NOP56, and the U3-specific protein Rrp9p/U3-55K. Snu13p/SNU13 has a much lower affinity for U3(C'/D) but nevertheless forms on this motif an RNP with box C/D proteins Nop1p/FBL and Nop58p/NOP58. In this study, we characterized the influence of the RNP assembly protein Rsa1 in the early steps of U3 snoRNP biogenesis in yeast and we propose a refined model of U3 snoRNP biogenesis. While recombinant Snu13p enhances the binding of Rrp9p to U3(B/C), we observed that Rsa1p has no effect on this activity but forms with Snu13p and Rrp9p a U3(B/C) pre-RNP. In contrast, we found that Rsa1p enhances Snu13p binding on U3(C'/D). RNA footprinting experiments indicate that this positive effect most likely occurs by direct contacts of Rsa1p with the U3 snoRNA 5' domain. In light of the recent U3 snoRNP cryo-EM structures, our data suggest that Rsa1p has a dual role by also preventing formation of a premature functional U3 RNP.