The objective of this study was to examine the long-term effects of different levels of dietary carbohydrates on glucose metabolism in the omnivorous Nile Tilapia (Oreochromis niloticus). Fish were fed from the first feeding stage until the adult stage, over a period of 40 weeks, using three different levels of carbohydrates (dextrin): 0% (CHO-L), 30% (CHO-M) and 50% (CHO-H). Growth performance, blood metabolite parameters, the proximate composition of the whole body, muscle and liver tissue, and the mRNA levels for genes involved in glycolysis, gluconeogenesis, lipogenesis and glucose transport in liver and muscle tissue were analyzed at week 26 and week 40. Fish fed the CHO-M diet exhibited the best growth performance, while the fish fed with the CHO-H diet showed the lowest growth performance. These data suggest that the incorporation of a certain level dietary carbohydrates can benefit Nile Tilapia. However, if the proportion of carbohydrates is very high, and concomitantly, the level of dietary proteins too low, this will not promote growth. Fish fed with carbohydrates exhibited a significant increase in lipid deposition in the whole body, muscle and liver tissues in. This seems to be due to increased lipogenic capacity, as reflected by higher hepatic fatty acid synthase mRNA levels and activity, as well as higher levels of plasma triglyceride. Nile Tilapia appear to be able to effectively adapt to the intake of carbohydrates, as reflected by: (i) the weak postprandial hyperglycemia (5.3–6.1 mM), (ii) the higher level of hepatic glycogen, (iii) the higher glycolytic pyruvate kinase enzyme in muscle (mRNA level and activity) and (iv) the inhibition of the gluconeogenic pathway for glucose-6-phosphatase and phosphoenolpyruvate carboxykinase enzymes (at molecular and enzymatic levels). Overall, these data show that the well-known differences in the capacity of carnivorous and omnivorous fish species to use dietary carbohydrates could be linked to differences in the regulation of the gluconeogenic and lipogenic pathways.