The human pathogen Neisseria meningitidis is the major causative agent of bacterial meningitis. The organism is usually treated as a strict aerobe and is cultured under fully aerobic conditions in the laboratory. We demonstrate here that although N. meningitidis fails to grow under strictly anaerobic conditions, under oxygen limitation the bacterium expresses a denitrification pathway (reduction of nitrite to nitrous oxide via nitric oxide) and that this pathway supplements growth. The expression of the gene aniA, which encodes nitrite reductase, is regulated by oxygen depletion and nitrite availability via transcriptional regulator FNR and two-component sensor-regulator NarQ/NarP respectively. Completion of the two-step denitrification pathway requires nitric oxide (NO) reduction, which proceeds after NO has accumulated during batch growth under oxygen-limited conditions. During periods of NO accumulation both nitrite and NO reduction are observed aerobically, indicating N. meningitidis can act as an aerobic denitrifier. However, under steady-state conditions in which NO is maintained at a low concentration, oxygen respiration is favoured over denitrification. NO inhibits oxidase activity in N. meningitidis with an apparent Ki NO = 380 nM measured in intact cells. The high respiratory flux to nitrite after microaerobic growth and the finding that accumulation of the denitrification intermediate NO inhibits oxygen respiration support the view that denitrification is a pathway of major importance in N. meningitidis.