Cells must make careful use of the resources available to them. A key area of cellular regulation involves the biogenesis of ribosomes. Transcriptional regulation of ribosome biogenesis factor genes through alterations in histone acetylation has been well studied. This work identifies a post-transcriptional mechanism of ribosome biogenesis regulation by Puf protein control of mRNA stability. Puf proteins are eukaryotic mRNA binding proteins that play regulatory roles in mRNA degradation and translation via association with specific conserved elements in the 3ʹ untranslated region (UTR) of target mRNAs and with degradation and translation factors. We demonstrate that several ribosome biogenesis factor mRNAs in Saccharomyces cerevisiae containing a canonical Puf4p element in their 3ʹ UTRs are destabilized by Puf2p, Puf4, and Puf5p, yet stabilized by Puf1p and Puf3p. In the absence of all Puf proteins, these ribosome biogenesis mRNAs are destabilized by a secondary mechanism involving the same 3ʹ UTR element. Unlike other targets of Puf4p regulation, the decay of these transcripts is not altered by carbon source. Overexpression of Puf4p results in delayed ribosomal RNA processing and altered ribosomal subunit trafficking. These results represent a novel role for Puf proteins in yeast as regulators of ribosome biogenesis transcript stability.