The bacterial oxidation of nitrite to nitrate is a key process of the biogeochemical nitrogen cycle. Nitrite-oxidizing bacteria are considered a highly specialized functional group, which depends on the supply of nitrite from other microorganisms and whose distribution strictly correlates with nitrification in the environment and in wastewater treatment plants. On the basis of genomics, physiological experiments, and single-cell analyses, we show that Nitrospira moscoviensis, which represents a widely distributed lineage of nitrite-oxidizing bacteria, has the genetic inventory to utilize hydrogen (H-2) as an alternative energy source for aerobic respiration and grows on H-2 without nitrite. CO2 fixation occurred with H-2 as the sole electron donor. Our results demonstrate a chemolithoautotrophic lifestyle of nitrite-oxidizing bacteria outside the nitrogen cycle, suggesting greater ecological flexibility than previously assumed.