The role of nucleic acids in prion aggregation / disaggregation has remained unclear. Here, using HET-s prion from fungi Podospora anserina as a model system, we have studied the role of ribosomal RNA (rRNA) in its aggregation process in an unbiased manner. Our results show that rRNA, in particular domain V of rRNA from the large subunit of the ribosome, substantially prevents beta-amyloid aggregation of the HET-s prion in a concentration dependent manner. The sites of interaction of the HET-s prion on domain V rRNA have been identified, which overlap with the sites previously identified for the protein folding activity of the ribosome. This study provides a missing link for the role of rRNA based folding activity of the ribosome in the context of prion propagation.