Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia. In plants, it occurs as two major isoforms, a cytosolic form (GS1) and a nuclear encoded chloroplastic form. The focus of this paper is to determine the role of the 5′UTR of a GS1 gene. GS1 gene constructs with and without its 5′ and 3′ UTRs, driven by a constitutive promoter, were agroinfiltrated into tobacco leaves and the tissues were analyzed for both transgene transcript and protein accumulation. The constructs were also tested in an in vitro transcription/translation system and in Escherichia coli. Our results showed that while the 3′ UTR functioned in the destabilization of the transcript, the 5′ UTR acted as a translation enhancer in plant cells but not in the in vitro translation system. The 5′UTR of the GS1 gene when placed in front of a reporter gene (uidA), showed a 20-fold increase in the level of GUS expression in agroinfiltrated leaves when compared to the same gene construct without the 5′UTR. The 5′UTR-mediated translational enhancement is probably another step in the regulation of GS in plants. The presence of the GS1 5′ UTR in front of the GS1 coding region allowed for its translation in E. coli suggesting the commonality of the translation initiation mechanism for this gene between plants and bacteria.