We combined genetic selection and dietary treatment to produce a model to study metabolic pathways involved in genetic and nutritional control of fat deposition in fish muscle. Two experimental lines of rainbow trout, selected for a lean (L) or fat (F) muscle, were fed with diets containing either 10 or 23% lipids from the first feeding, up to 6 mo. At the end of the feeding trial, trout were distinguished by very different muscle fat content (from 4.2 to 10% wet weight), and line x diet interactions were observed for parameters related to fat storage. We analyzed the activity and gene expression of key enzymes involved in lipid metabolism (fatty acid synthase, hydroxyacyl-CoA dehydrogenase, carnitine palmitoyltransferase 1 isoforms, and peroxisome proliferator-activated receptor alpha) and glycolysis (hexokinase 1 and pyruvate kinase) as well as energy production (isocitrate dehydrogenase, citrate synthase, and cytochrome oxidase) in the liver and the white muscle of rainbow trout. The lipid-rich diet repressed the activity of the lipogenic enzymes and stimulated enzymes involved in fatty acid oxidation and glycolysis in liver but had little effect on muscle enzymes assessed in this study. Regarding the selection effect, enzyme activity and expression suggest that compared with the L line, the F line presented reduced hepatic fatty acid oxidation as well as reduced mitochondrial oxidative capacities and enhanced glucose utilization in both liver and muscle. Very few line x diet interactions were found, suggesting that the two factors (i.e., dietary energy content and selection) used in this study to modify muscle lipid content exerted some additive but mostly independent effects on these metabolic actors.