Updated project metadata. The nitrogen rich compound guanidine occurs widely in nature and is used by microbes as a nitrogen source, but microorganisms that grow on guanidine have not yet been discovered. Here we show that complete ammonia-oxidizing microbes (comammox), but no other known nitrifiers, encode homologues of a guanidinase and that the comammox isolate Nitrospira inopinata grows on guanidine as sole source of energy and reductant. Proteomics, kinetic enzyme characterization, and the crystal structure of the N. inopinata guanidinase homologue demonstrated that it is a bona fide guanidinase. Transcription of comammox guanidinases was induced in wastewater treatment plant microbiomes upon incubation with guanidine, and guanidine degradation was detected in these systems. The discovery of guanidine as a selective growth substrate for comammox shows a unique niche of these globally important nitrifiers and offers new options for their isolation as well as for targeted manipulation of nitrifier communities.