Abstract Molecular hydrogen (H 2 ) is among the most central, but least understood, metabolites in the human gastrointestinal tract (gut). H 2 gas is produced in large quantities during bacterial fermentation and consumed as an energy source by bacteria and archaea. Disruption of H 2 cycling is linked to gastrointestinal disorders, infections, and cancers, with H 2 used as an indicator of gut dysfunction through breath tests. Despite this, the microorganisms, pathways, and enzymes mediating H 2 production remain unresolved. Here we show that a previously uncharacterised enzyme, the group B [FeFe]-hydrogenase, drives most fermentative H 2 production in the human gut. Analysis of stool, biopsy, and isolate (meta)genomes and (meta)transcriptomes show this hydrogenase is encoded by most gut bacteria and is highly expressed. Through analysis of 19 taxonomically diverse gut isolates, the group B [FeFe]-hydrogenase produces large amounts of H 2 gas and supports fermentative growth of both Bacteroidetes and Firmicutes. Bacteroides particularly dominate H 2 production. Biochemical and spectroscopic characterisation shows purified group B [FeFe]-hydrogenases are catalytically active and bind a di-iron active site. These hydrogenases are highly enriched in the guts of healthy individuals, but significantly depleted in favour of other fermentative hydrogenases in Crohn’s disease. Furthermore, we show that metabolically flexible respiratory bacteria are the most abundant H 2 oxidizers in the gut, not sulfate reducers, methanogens, and acetogens as previously thought. This combination of enzymatic, cellular, and ecosystem-level analysis provides the first detailed understanding of H 2 cycling in the human gut and reveals new links between microbiota function and gastrointestinal health.

A widespread hydrogenase drives fermentative growth of gut bacteria in healthy people