Anaerobic ammonium-oxidation (anammox) reaction is a crucial microbial nitrogen (N) transformation process contributing to the global N cycling. Only two families and six candidate genera of anammox bacteria have been described in the past two decades. It remains unclear whether the currently discovered anammox lineages can fully represent all potential anammox bacterial diversity. Here, we discovered and named a novel anammox bacterial order, Candidatus Aquiferales. A high-quality metagenome-assembled genome of this order, tentatively named “Ca. Mazuia houmensis JP1”, was recovered from a coastal aquifer adjacent to the South China Sea. Genome of “Ca. Mazuia houmensis JP1” not only contains genes encoding the key enzymes of the anammox metabolism (e.g., hydrazine synthase, hydrazine oxidoreductase, nitrite reductase, and nitrite oxidoreductase) but also has diverse oxygen resistance strategies. The new oxygen resistance pathway of the novel cholesterol oxidase coupled with cytochrome c peroxidase may confer a selective advantage for these new anammox bacteria to adapt the changing redox condition in the coastal aquifer. Furthermore, comparatively smaller genome size (<2.5 Mbp) and higher coding density (>85%) likely confer another selective advantage for them in the oligotrophic aquifer. 15N isotope tracing combined with amplicon sequencing and q-PCR analysis of anammox specific-16S rRNA gene showed that the saltwater–freshwater interface in coastal aquifer is a favorable niche for these novel anammox bacteria. The study would largely expand our knowledge of the taxonomic and metabolic diversity of anammox bacteria and reveal the ecological function of anammox process in the nitrogen cycle at the land-sea interface.