SummaryBacteroidetes VC2.1 Bac22 (referred to as VC2.1) is an uncultured clade that is widely distributed in marine ecosystems, including hydrothermal vents, oxygen‐minimum zones and other anoxic, sulfide‐rich environments. However, the lack of cultured representatives and sequenced genomes of VC2.1 limit our understanding of its physiology, metabolism and ecological functions. Here, we obtained a stable co‐culture of VC2.1 with autotrophic microbes by establishing an autotrophy‐based enrichment from a hydrothermal vent chimney sample. We recovered a high‐quality metagenome‐assembled genome (MAG) that belonged to VC2.1. Phylogenetic analyses of both 16S rRNA genes and conserved protein markers suggested that VC2.1 belongs to a novel order in the Bacteroidetes phylum, which we named Candidatus Sulfidibacteriales. The metabolic reconstruction of this MAG indicated that VC2.1 could utilize polysaccharides, protein polymers and fatty acids as well as flexibly obtain energy via NO/N₂O reduction and polysulfide reduction. Our results reveal the ecological potential of this novel Bacteroidetes for complex organic carbons mineralization and N₂O sinks in deep‐sea hydrothermal vents. Furthermore, guided by the genome information, we designed a new culture medium in which starch, ammonium and polysulfide were used as the carbon source, nitrogen source and electron acceptor respectively, to isolate VC2.1 successfully.