Marine sediments represent the largest reservoir of methane on Earth. Anaerobic oxidation of methane (AOM) mediated by anaerobic methanotrophic archaea (ANME) and associated sulfate-reducing bacteria is the main process controlling methane emission from marine sediments. Recent laboratory studies have revealed that ANME or AOM consortia can mediate nitrogen (N) fixation and ammonium assimilation. These processes are known to preferentially utilize 14N rather than the 15N isotope. However, geochemical fingerprints of microbial nitrogen uptake in marine sediments are lacking, limiting our understanding of its role in modern and ancient sedimentary environments. Here we report prominent negative excursions of bulk sediment δ15N values (up to −5‰) at sulfate-methane transitions (SMTs) where ANME show peak abundances in sediments at four sites of the Haima seeps of the South China Sea. Such δ15N excursions are restricted to SMTs, suggesting that 15N depletion results from ANME-mediated nitrogen uptake. We posit that this process is ubiquitous at the SMT – a reaction front in coastal and continental margin sediments worldwide – resulting in similar isotope patterns. Negative δ15N excursions are consequently a promising proxy to track AOM in the geological record and to better constrain carbon and nitrogen cycling in ancient sedimentary environments.