Alanine aminotransferase (ALT) catalyses a transamination reaction that links carbohydrate and amino acid metabolism. In this study, we examined the effect of silencing cytosolic ALT (cALT) expression on the hepatic metabolism in Sparus aurata. A number of siRNA and shRNA designed to down-regulate cALT expression were validated in HEK-293 cells transfected with plasmids expressing S. aurata cALT or mitochondrial ALT (mALT) isoforms: ALT silencing significantly decreased the expression levels of S. aurata mRNA cALT1 to 62% (siRNA) and 48% (shRNA) of the values observed in control cells. The effect of cALT silencing was analysed in the liver of S. aurata 72 h after intraperitoneal injection of chitosan-tripolyphosphate (TPP) nanoparticles complexed with a plasmid encoding a shRNA to down-regulate cALT expression (pCpG-si1sh1). In fish fed diets with different ratio of protein to carbohydrate and treated with chitosan-TPP-pCpG-si1sh1, cALT1 and cALT2 mRNA levels significantly decreased irrespective of the diet. Consistently, ALT activity decreased in liver of treated animals. In the liver of S. aurata treated with chitosan-TPP-pCpG-si1sh1 nanoparticles, down-regulation of cALT expression increased the activity of key enzymes in glycolysis (6-phosphofructo-1-kinase and pyruvate kinase) and protein metabolism (glutamate dehydrogenase). Besides showing for the first time that administration of chitosan-TPP-pCpG-si1sh1 nanoparticles silences hepatic cALT expression in vivo, our data support that down-regulation of cALT could improve the use of dietary carbohydrates to obtain energy and spare protein catabolism.