SummaryStimulating litho‐autotrophic denitrification in aquifers with hydrogen is a promising strategy to remove excess NO₃⁻, but it often entails accumulation of the cytotoxic intermediate NO₂⁻ and the greenhouse gas N₂O. To explore if these high NO₂⁻ and N₂O concentrations are caused by differences in the genomic composition, the regulation of gene transcription or the kinetics of the reductases involved, we isolated hydrogenotrophic denitrifiers from a polluted aquifer, performed whole‐genome sequencing and investigated their phenotypes. We therefore assessed the kinetics of NO₂⁻, NO, N₂O, N₂ and O₂ as they depleted O₂ and transitioned to denitrification with NO₃⁻ as the only electron acceptor and hydrogen as the electron donor. Isolates with a complete denitrification pathway, although differing intermediate accumulation, were closely related to Dechloromonas denitrificans, Ferribacterium limneticum or Hydrogenophaga taeniospiralis. High NO₂⁻ accumulation was associated with the reductases' kinetics. While available, electrons only flowed towards NO₃⁻ in the narG‐containing H. taeniospiralis but flowed concurrently to all denitrification intermediates in the napA‐containing D. denitrificans and F. limneticum. The denitrification regulator RegAB, present in the napA strains, may further secure low intermediate accumulation. High N₂O accumulation only occurred during the transition to denitrification and is thus likely caused by delayed N₂O reductase expression.