Glucose intolerance in carnivorous fish has been attributed to the lack of hepatic glucokinase (GK) activity. Transcription/translation assay and transient transfection of COS-7 cells with a cDNA encoding Sparus aurata liver GK showed the functionality of the enzyme in vitro. The endogenous fish hepatic GK had lower affinity for glucose than the rat enzyme. The GK activity values in fed fish were similar to those reported for starved and diabetic rats. In this study, we also addressed the nutritional regulation of GK gene expression in fish liver. Starvation and energy restriction decreased S. aurata hepatic GK mRNA and activity levels, as previously reported in rats. In contrast, the fish enzyme expression exhibited a delayed onset during the daily feeding rhythm. These findings demonstrate for the first time the presence and the nutritional modulation of a functional GK activity in fish liver and contribute to explain the low ability of carnivorous fish to metabolize carbohydrates.