[NiFe]‐hydrogenase (Hyd) 2 of Escherichia coli has been proposed to generate proton motive force during H2‐oxidation, which it is dependent on if cells are incubated anaerobically with glycerol to drive reverse H2‐production. The integral membrane subunit HybB is required for proton transfer (PT) by Hyd‐2 but has no cofactor. To provide evidence for PT by HybB, we analyzed the roles of conserved amino acid residues in a predicted proton channel. Exchange of conserved residues identified residues Y99, E133, H184, and E228 as mandatory for PT from the cytoplasm and quinol oxidation. In contrast, exchange of W54, D58, or R89 rendered Hyd‐2 uni‐directional and influenced the equilibrium. Our findings show that HybB is the key subunit in PT.