To contribute to the understanding of the mechanisms involved in poor metabolic utilization of dietary carbohydrates by rainbow trout (Oncorhynchus mykiss), we explored in this study the effects of dietary lipids on the regulation of two hepatic key enzymes, i.e., glucokinase (GK, first enzyme of the glycolytic pathway) and glucose-6-phosphatase (G6Pase, last enzyme of the gluconeogenesis). Two groups of juvenile trout were pair-fed for 8 wk either a low (10%) or a high (25%) level of dietary lipids supplied as fish oil; the pair-feeding technique was adopted to vary fat intake while keeping the protein and carbohydrate intakes more or less constant. Fish fed the high level of dietary lipids had inefficient control of glycemia compared with fish fed the low level of lipids. Levels of dietary lipids did not affect GK activity even though there was a small increase of GK mRNA level at 3 h after feeding high levels of lipids. By contrast, the high level of dietary lipids significantly increased G6Pase mRNA expression at 3, 6 and 12 h and enzyme activity at 6 h after food consumption. Thus, these data suggest that poor dietary carbohydrate utilization in rainbow trout may be related at least in part to increased hepatic glucose production under conditions of high dietary fat intake.