In order to reconstruct the ecosystem structure of chemosynthetic environments in the fossil record, geochemical proxies must be developed. Here, we present a suite of novel compound-specific isotope parameters for tracing chemosynthetic production with a focus on understanding nitrogen dynamics in deep-sea cold seep environments. We examined the chemosymbiotic bivalve Bathymodiolus childressi from three geographically distinct seep sites on the NE Atlantic Margin and compared isotope data to non-chemosynthetic littoral mussels to test whether water depth, seep activity, and/or mussel bed size are linked to differences in chemosynthetic production. The bulk isotope analysis of carbon (δ13C) and nitrogen (δ15N), and δ15N values of individual amino acids (δ15NAA) in both gill and muscle tissues, as well as δ15NAA-derived parameters including trophic level (TL), baseline δ15N value (δ15NPhe), and a microbial resynthesis index (ΣV), were used to investigate specific geochemical signatures of chemosynthesis. Our results show that δ15NAA values provide a number of new proxies for relative reliance on chemosynthesis, including TL, ∑V, and both δ15N values and molar percentages (Gly/Glu mol% index) of specific AA. Together, these parameters suggested that relative chemoautotrophy is linked to both degree of venting from seeps and mussel bed size. Finally, we tested a Bayesian mixing model using diagnostic AA δ15N values, showing that percent contribution of chemoautotrophic versus heterotrophic production to seep mussel nutrition can be directly estimated from δ15NAA values. Our results demonstrate that δ15NAA analysis can provide a new set of geochemical tools to better understand mixotrophic ecosystem function and energetics, and suggest extension to the study of ancient chemosynthetic environments in the fossil record.