Abstract: Phosphoenolpyruvate carboxykinase (PEPCK, E.C.4.1.1.32) is a key gluconeogenic enzyme in aquatic organisms. In this experiment, three marine fish species with different food habits -omnivorous Tilapia (Oreochromis niloticus), moderate carnivorous Derbio (Trachinotus ovatus) and ferocious carnivorous Cobia (Rachycentron canadum) were selected as the experimental subjects. Dextrin was used as the main dietary carbohydrate source to generate three equal-nitrogen groups of diets with different carbohydrate levels (low dextrin LD, middle dextrin MD and high dextrin HD). Sixty juvenile fish with normal size were randomly selected from each species and fed for 8 weeks during the experimental period. To identify the effects of dietary carbohydrate levels on the gene expression and the enzymatic acidity of PEPCK, we also cloned and analyzed the full-length sequence of PEPCK gene cDNA of Trachinotus ovatus. The full-length cDNA of Derbio PEPCK was 2652 bp and encoded 624 amino acids. The bioinformatics study showed that PEPCK gene was highly conserved between the three species and the homology was higher than 90%. The PEPCK activity of Derbio and Cobia were reduced by 28.05% and 26.03% respectively in the HD groups compared to the LD groups (P0.05), suggesting the increased carbohydrate level may cause decrease in the enzymatic activity. The expression of PEPCK in the liver of Tilapia, Derbio and Cobia was also reduced with the increase in the carbohydrate level. Compared to the LD group, the PEPCK mRNA level of the HD group exhibited a 100-, 4.3-and 4.77-fold decrease in Tilapia, Derbio and Cobia respectively. These results suggested that the gluconeogenic capacity might be related to the food habit of the fish, and that the gene expression and the enzymatic activity of PEPCK of the three fish species could be significantly inhibited by the elevated dietary dextrin level. The degree of inhibition in mRNA expression also varied probably due to different food habits, and the highest inhibition appeared in Tilapia and the lowest in Cobia.