Some fish show a low metabolic ability to use dietary carbohydrates. The use of early nutritional stimuli to program metabolic pathways in fish is ill defined. Therefore, studies were undertaken with zebrafish to assess the effect of high glucose levels during the embryonic stage as a lifelong modulator of genes involved in carbohydrate metabolism. Genes related to carbohydrate metabolism were expressed at low levels at 0.2 and 1 day post-fertilization (dpf). However, from 4 dpf onwards there was a significant increase on expression of all genes, suggesting that all analysed pathways were active. By microinjection, we successfully enriched zebrafish egg yolk with glucose (a 43-fold increase of basal levels). Acute effects of glucose injection on gene expression were assessed in larvae up to 10 dpf, and the programming concept was evaluated in juveniles (41 dpf) challenged with a hyperglucidic diet. At 4 dpf, larvae from glucose-enriched eggs showed a downregulation of several genes related to glycolysis, glycogenolysis, lipogenesis and carbohydrate digestion in comparison with control (saline-injected) embryos. This inhibitory regulation was suppressed after 10 dpf. At the juvenile stage, and upon switching from a low to a high digestible carbohydrate diet, early glucose enrichment had no significant effect on most analysed genes. However, these same fish showed altered expression of the genes for cytosolic phosphoenolpyruvate carboxykinase, sodium-dependent glucose cotransporter 1 and glycogen synthase, suggesting changes to the glucose storage capacity in muscle and glucose production and transport in viscera. Overall, supplementation of egg yolk with high glucose levels had little effect on the long-term modulation of carbohydrate metabolic genes in zebrafish