The human Y-box protein 1 (YB-1) is a member of the Y-box protein family, a class of proteins involved in transcriptional and translational regulation of a wide range of genes. Here, we report the solution structure of the cold-shock domain (CSD) of YB-1, which is thought to be responsible for nucleic acid binding. It is the first structure solved of a eukaryotic member of the cold-shock protein family and consists of a closed five-stranded anti-parallel β-barrel capped by a long flexible loop. The structure of CSD is similar to the OB-fold and a comparison with bacterial cold-shock proteins shows that its structural properties are conserved from bacteria to man. Our data suggest the presence of a DNA-binding site consisting of a patch of positively charged and aromatic residues on the surface of the β-barrel. Further, it is shown that CSD, which has a preference for binding single-stranded pyrimidine-rich sequences, binds weakly and hardly specifically to DNA. Binding affinities reported for intact YB-1 indicate that domains other than the CSD play a role in DNA binding of YB-1.