Glutamate dehydrogenase (Gdh) plays a central role in ammonia detoxification by catalysing reversible oxidative deamination ofl-glutamate into α-ketoglutarate using NAD⁺or NADP⁺as cofactor. To gain insight into transcriptional regulation ofglud, the gene that codes for Gdh, we isolated and characterised the 5′ flanking region ofgludfrom gilthead sea bream (Sparus aurata). In addition, tissue distribution, the effect of starvation as well as short- and long-term refeeding on Gdh mRNA levels in the liver ofS. auratawere also addressed. 5′-Deletion analysis ofgludpromoter in transiently transfected HepG2 cells, electrophoretic mobility shift assays, chromatin immunoprecipitation (ChIP) and site-directed mutagenesis allowed us to identify upstream stimulatory factor 2 (Usf2) as a novel factor involved in the transcriptional regulation ofglud. Analysis of tissue distribution of Gdh and Usf2 mRNA levels by reverse transcriptase-coupled quantitative real-time PCR (RT-qPCR) showed that Gdh is mainly expressed in the liver ofS. aurata, while Usf2 displayed ubiquitous distribution. RT-qPCR and ChIP assays revealed that long-term starvation down-regulated the hepatic expression of Gdh and Usf2 to similar levels and reduced Usf2 binding togludpromoter, while refeeding resulted in a slow but gradual restoration of both Gdh and Usf2 mRNA abundance. Herein, we demonstrate that Usf2 transactivatesS. aurata gludby binding to an E-box located in the proximal region ofgludpromoter. In addition, our findings provide evidence for a new regulatory mechanism involving Usf2 as a key factor in the nutritional regulation ofgludtranscription in the fish liver.