Background Stable isotope analysis of single amino acids (AA) is usually applied in food web studies for tracing biosynthetic origins of AA carbon backbones and establishing trophic positions of consumers, but the method is also showing promise for characterizing quantity and quality of dietary lipids and carbohydrates. Methods To investigate whether changes in high- and low-digestible carbohydrates affect δ 13 C values of glycolytic AA, i.e., AA carbon backbones sourced from the glycolytic pathway, we compared Atlantic salmon ( Salmo salar ) from a feeding experiment with and without dietary inclusion of the red macroalga Palmaria palmata . The Control and experimental diets had similar relative proportions of macronutrients, but their ingredients differed; in the experimental treatment, 15% Palmaria inclusion substituted proteins from fishmeal and carbohydrates from corn starch. Results We found that 13 C values of the glycolytic AA were highly sensitive to substitution of corn starch with Palmaria . The δ 13 C offsets of glycolytic AA between salmon and their diets were significantly greater in the Palmaria inclusion than Control treatment. This greater offset can be attributed to the different utilization of high- vs. low-digestible carbohydrate sources, i.e., corn starch vs. Palmaria , in the two treatments, and metabolic routing of dietary lipids. In addition, similar δ 13 C values of essential AA between treatments indicate similar nutrient assimilation efficiency for all terrestrial (pea protein concentrate and wheat gluten meal) and marine (fishmeal and red alga) derived protein sources. These results show that δ 13 C AA analysis is a promising tool for improving our understanding of how carnivorous fish utilize macronutrient and route metabolic intermediates to tissue.