Here, we successfully used NO as the direct electron acceptor for the enrichment of a microbial community in a continuous bioreactor. The enrichment culture, mainly comprised of two new organisms from the Sterolibacteriaceae family, grew on NO reduction to N2 and formate oxidation, with virtually no accumulation of N2O. The microbial growth kinetics of the enrichment culture as well as its affinity for different N-oxides were determined. In parallel, using metagenomics, metatranscriptomics, and metaproteomics, the biochemical reactions underlying the growth of these microorganisms on NO were investigated. This study demonstrates that microorganisms thrive and can be enriched on NO, and presents new opportunities to study microbial growth on this highly energetic and climate-active molecule that may have been pivotal in the evolution of aerobic respiration.