mRNA stability is elaborately regulated by elements in the mRNA transcripts and their cognate RNA-binding proteins, which play important roles in regulating gene expression at the post-transcriptional level in eukaryotes. Poly(U)-binding protein 1 (Pub1), which is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae, is involved in the regulation of mRNA turnover as a trans-acting factor. It binds to transcripts containing the AU-rich element in order to protect them from degradation. Pub1 contains three RNA-recognition motifs (RRMs) which play significant roles in mRNA binding at AU-rich elements and stabilizer elements. In this study, the second RRM of Pub1 was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 4000 as a precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group H3.