High levels of dietary lipids are incorporated in feeds for most teleost fish to promote growth and reduce nitrogen waste. However, in Senegalese sole (Solea senegalensis) previous studies revealed that increasing the level of dietary lipids above 8% negatively affect growth and nutrient utilization regardless of dietary protein content. It has been shown that glucose regulation and metabolism can be impaired by high dietary fat intake in mammals, but information in teleost fish is scarce. The aim of this study was to assess the possible effect of dietary lipids on glucose metabolism in Senegalese sole with special emphasis on the regulation of proteins involved in the muscle insulin-signalling pathway. Senegalese sole juveniles (29 g) were fed two isonitrogenous diets (53% dry matter) for 88 days. These two diets were one with a high lipid level (∼17%, HL) and a moderate starch content (∼14%, LC), and the other being devoid of fish oil (4% lipid, LL) and with high starch content (∼23%, HC). Surprisingly, feeding Senegalese sole the HL/LC diet resulted in prolonged hyperglycaemia, while fish fed on LL/HC diet restored basal glycaemia 2 h after feeding. The hyperglycaemic phenotype was associated with greater glucose-6-phosphatase activity (a key enzyme of hepatic glucose production) and lower citrate synthase activity in the liver, with significantly higher liver glycogen content. Sole fed on HL/LC diet also had significantly lower hexokinase activity in muscle, although hexokinase activity was low with both dietary treatments. The HL/LC diet was associated with significant reductions in muscle AKT, p70 ribosomal S6-K1 Kinase (S6K-1) and ribosomal protein S6 (S6) 2 h after feeding, suggesting down regulation of the AKT-mTOR nutrient signalling pathway in these fish. The results of this study show for the first time that high level of dietary lipids strongly affects glucose metabolism in Senegalese sole.