In the present study, the effects of partial substitution of dietary protein by digestible starch on endogenous glucose production were evaluated in European seabass (Dicentrarchus labrax). The fractional contribution of dietary carbohydratesv.gluconeogenesis to blood glucose appearance and hepatic glycogen synthesis was quantified in two groups of seabass fed with a diet containing 30 % digestible starch (DS) or without a carbohydrate supplement as the control (CTRL). Measurements were performed by transferring the fish to a tank containing water enriched with 5 %²H₂O over the last six feeding days, and quantifying the incorporation of²H into blood glucose and hepatic glycogen by²H NMR. For CTRL fish, gluconeogenesis accounted for the majority of circulating glucose while for the DS fish, this contribution was significantly lower (CTRL 85 (sem4) %v.DS 54 (sem2) %;P< 0·001). Hepatic glycogen synthesis via gluconeogenesis (indirect pathway) was also significantly reduced in the DS fish, in both relative (CTRL 100 (sem1) %v.DS 72 (sem1) %;P< 0·001) and absolute terms (CTRL 28 (sem1)v.DS 17 (sem1) μmol/kg per h;P< 0·001). A major fraction of the dietary carbohydrates that contributed to blood glucose appearance (33 (sem1) % of the total 47 (sem2) %) had undergone exchange with hepatic glucose 6-phosphate. This indicated the simultaneous activity of hepatic glucokinase and glucose 6-phosphatase. In conclusion, supplementation of digestible starch resulted in a significant reduction of gluconeogenic contributions to systemic glucose appearance and hepatic glycogen synthesis.