The link between dietary carbohydrate/protein and de novo lipogenesis (DNL) remains debatable in carnivorous fish. We evaluate the response of hepatic lipogenic gene expression to dietary carbohydrate intake/glucose and dietary protein intake/amino acids (AAs) during acute stimulations in trout. For the in vivo trial, three different diets and a controlled-feeding method were employed to supply fixed amount of dietary protein or carbohydrate in a single meal; for the in vitro trial, primary hepatocytes were stimulated with low/high level of glucose (3 mM or 20 mM) and low/high level of AAs (one-fold or four-fold concentrated AAs). In vitro data showed that high level of AAs up-regulated the expression of enzymes involved in DNL (FAS and ACLY), lipid bioconversion (Elovl5, Elovl2, D6D and SCD1), NADPH production (G6PDH and ME), and transcriptional factor SREBP1c, while high level of glucose only elevated the expression of ME. Data in trout liver also showed that high dietary protein intake induced higher lipogenic gene expression (FAS, ACLY and Elovl2) regardless of dietary carbohydrate intake, while high carbohydrate intake markedly suppressed the expression of ACC and Elovl5. Overall, we conclude that, unlike rodents or humans, DNL gene expression in rainbow trout is more responsive to dietary protein intake/AAs than dietary carbohydrate intake/glucose during acute stimulations. This discrepancy probably represents one important physiological and metabolic difference between carnivores and omnivores.