Glutamine synthetase (GS) is a widely distributed enzyme in animals and plants, which is involved in various cellular activities. In crustaceans, GS plays a major role in the regulation of energy metabolism and osmotic pressure. In the present report, a full cDNA of the GS gene with 1 965 bp in length was obtained from giant freshwater prawn (Macrobrachium rosenbergii ). The open reading frame (ORF) encoded a 361 amino acid (aa) polypeptide with the predicted molecular weight of 40.75 ku. Analysis of amino acid sequences showed that the GS of M. rosenbergii contained 5 conserved regions that belong to the GS Ⅱ group of invertebrates. Phylogenetic analysis of GS of M. rosenbergii shared the highest identity within the clade of GS from Litopenaeus vannamei, Penaeus monodon, and Fenneropenaeus chinensis with 95% similarities at aa level. Polyclonal antibody against GS of M. rosenbergii was generated. Furthermore, we detected the expression profiles of GS mRNA and proteins in various tissues of M. rosenbergii by quantitative reverse transcription PCR (qRT-PCR) and Western blotting. GS mRNA was differentially expressed in 8 detected tissues of M. rosenbergii before molting in the order of hepatopancreas > muscle > stomach > intestine > gill > heart > brain > haemolymph. The expressions of GS in different tissues were compared before and after molting. The results showed that, except in hepatopancreas, the mRNA expression of GS was up-regulated in the other 7 tissues in the order of brain > gill > stomach > intestine > muscle > heart > haemolymph. By Western blot, the expressions of GS protein in the gill and muscles were up-regulated after molting, which was identical with the expression profiles at mRNA level. In addition, the activity of GS enzyme and the concentration of glutamine were decreased in hepatopancreas after molting, and by contrast, they were increased in the gill, muscle and haemolymph. These results were also identical with the GS mRNA expression. The differentiated expressions of GS gene in different tissues before and after molting suggest that it might be involved in the regulation of energy metabolism and osmotic pressure during the molting.