Beyond a few obvious examples (e.g., gut length, amylase activity), digestive and metabolic specializations towards diet remain elusive in fishes. Thus, we compared gut length, δ13C and δ15N signatures of the liver, and expressed genes in the intestine and liver of wild-caught individuals of four closely-related, sympatric prickleback species (family Stichaeidae) with different diets: Xiphister mucosus (herbivore), its sister taxon X. atropurpureus (omnivore), Phytichthys chirus (omnivore) and the carnivorous Anoplarchus purpurescens. We also measured the same parameters after feeding them carnivore or omnivore diets in the laboratory for 4 weeks. Growth and isotopic signatures showed assimilation of the laboratory diets, and gut length was significantly longer in X. mucosus in comparison to the other fishes, whether in the wild, or in the lab consuming the different diets. Dozens of genes relating to digestion and metabolism were observed to be under selection in the various species, but P. chirus stood out with some genes in the liver showing strong positive selection, and these genes correlating with differing isotopic incorporation of the laboratory carnivore diet in this species. Although the intestine showed variation in the expression of hundreds of genes in response to the laboratory diets, the liver exhibited species-specific gene expression patterns that changed very little (generally <40 genes changing expression, with P. chirus providing an exception). Overall, our results suggest that the intestine is plastic in function, but the liver may be where specialization manifests since this tissue shows species-specific gene expression patterns that match with natural diet.