The rainbow trout (Oncorhynchus mykiss) is considered to be a strictly carnivorous fish species that is metabolically adapted for high catabolism of proteins and low utilisation of dietary carbohydrates. This species consequently has a "glucose-intolerant" phenotype manifested by persistent hyperglycaemia when fed a high-carbohydrate diet. Gluconeogenesis in adult fish is also poorly, if ever, regulated by carbohydrates, suggesting that this metabolic pathway is involved in this specific phenotype. In this study, we hypothesised that the additional round of whole genome duplication occurring in trout (Ss4R) could represent a major evolutionary event leading to adaptive innovation via gene duplication and might provide new elements to enhance our understanding of gluconeogenesis and poor dietary carbohydrates use in this species. Our evolutionary analysis of gluconeogenic genes revealed that pck1, pck2, fbp1a and g6pca were retained as singletons after Ss4r while g6pcb1, g6pcb2 and fbp1b ohnologue pairs were maintained. For all genes, duplication may have led to sub- or neo-functionalisation. Expression profiles suggested that the gluconeogenesis pathway remained active in trout fed a no carbohydrate diet. When trout were fed a high-carbohydrate diet (30%), most of the gluconeogenic genes were non- or down-regulated, except for g6pbc2 ohnologues the RNA levels of which were surprisingly increased. This study demonstrated that Ss4R in trout involved adaptive innovation via gene duplication and via the outcome of the resulting ohnologues. Indeed, maintenance of ohnologuous g6pcb2 pair may contribute in a significant way to the "glucose-intolerant" phenotype of trout and may partially explain its poor use of dietary carbohydrates.