AbstractOceanic methane from global deep-sea sediment is largely consumed through microbially mediated sulfate-coupled oxidation, resulting in 13C-depleted cell biomass of anaerobic methanotrophic archaea (ANME). The general ecological importance of subseafloor ANME has been well recognized in the last two decades. However, the crucial biochemical pathways for the overall anaerobic oxidation of methane (AOM) still remain enigmatic. Here, methanotrophic pathways were analyzed to trace 13C-depleted amino acid biosynthesis in two clades of ANME (ANME-1 and ANME-2) from the Black Sea. Compound-specific analysis of ANME-dominated microbial mats showed a significant 13C-depletion trend in association with increasing carbon numbers in protein-derived amino acid families (e.g., the pyruvate family in the order of alanine, valine, isoleucine and leucine was down to −114‰). This result indicates a stepwise elongation of 13C-depleted carbon during amino acid biosynthesis. The overall results suggest that intracellular protein amino acids and the most 13C-depleted signature of leucine, which has a specific branched-chain structure, are potentially propagated as isoprenoid precursor molecules into archaeal biosynthesis, resulting in the extremely 13C- and 14C-depleted nature of ANME cells in the deep microbial oasis.