The potential for microbial nitrogen fixation in the anoxic methane seep sediments in a mud volcano, the number 8 Kumano Knoll, was characterized by molecular phylogenetic analyses. A total of 111 of the nifH (a gene coding a nitrogen fixation enzyme, Fe protein) clones were obtained from different depths of the core sediments, and the phylogenetic analysis of the clones indicated the genetic diversity of nifH genes. The predominant group detected (methane seep group 2), representing 74% of clonal abundance, was phylogenetically related to the nifH sequences obtained from the Methanosarcina species but was most closely related to the nifH sequences potentially derived from the anoxic methanotrophic archaea (ANME-2 archaea). The recovery of the nif gene clusters including the nifH sequences of the methane seep group 2 and the subsequent reverse transcription-PCR detection of the nifD and nifH genes strongly suggested that the genetic components of the gene clusters would be operative for the in situ assimilation of molecular nitrogen (N(2)) by the host microorganisms. DNA-based quantitative PCR of the archaeal 16S rRNA gene, the group-specific mcrA (a gene encoding the methyl-coenzyme M reductase alpha subunit) gene, and the nifD and nifH genes demonstrated the similar distribution patterns of the archaeal 16S rRNA gene, the mcrA groups c-d and e, and the nifD and nifH genes through the core sediments. These results supported the idea that the anoxic methanotrophic archaea ANME-2c could be the microorganisms hosting the nif gene clusters and could play an important role in not only the in situ carbon (methane) cycle but also the nitrogen cycle in subseafloor sediments.