The Y-box protein family is highly conserved nucleic acid binding proteins which are conserved from bacteria to human. In this study, the cDNA encoding cold shock protein Y-box from oriental river pawn Macrobrachium nipponense (MnY-box) was cloned by using EST analysis and rapid amplification of cDNA ends (RACE) approach. The full length cDNA of MnY-box was 1501 bp, consisting of a 5' untranslated region of 84 bp, a 3' untranslated region of 541 bp, and an open reading frame of 876 bp. The deduced protein had 291 amino acid residues with molecular mass of 31.48 kDa. Based on the protein similarity comparison, the Y-box protein family was very conservative in CSD domain. Phylogenetic tree analysis stated that oriental river prawn has the closest relationship with Daphnia pulex. Quantitative real-time RT-PCR analysis showed that the MnY-box gene was expressed in haemocytes, hepatopancreas, muscles, gill, brain, testis, ovary and intestines with the highest level of expression in the hepatopancreas. After environmental low temperature and recovery challenge, the relative expression level of MnY-box in hepatopancreas was significantly higher compared to the control group at 3 h to 12 h post-low temperature, followed by return to control levels at recovery 12 h. What's more, we constructed prokaryotic expression system and obtained the recombinant proteins of M. nipponense Y-box, which may provide information for further functional study.