Although of potential biotechnological interest, photobiological H2 production from microalgae and cyanobacteria is strongly limited due to the oxygen sensitivity of hydrogenases, the H2-evolving enzymes. We study here the [NiFe] hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 to identify structural determinants of its sensitivity to O2. Based on previous work on the hydrogenase from Desulfovibrio fructosovorans and on a structural model of the Synechocystis hydrogenase, we have created various mutants of the Synechocystis enzyme. Amino acids residues homologous to those defining the end of the intramolecular gas channel in the D. fructosovorans enzyme were specifically targeted, these residues being previously described as critical for enzyme activity and tolerance to O2. We show here that mutation I64M of the Synechocystis enzyme alters gas diffusion kinetics and improves O2 tolerance. These results constitute the first report demonstrating that an O2 tolerance-related character could be transposed from a proteobacterial hydrogenase to a cyanobacterial one, and may constitute the first published improvement of O2 tolerance of a cyanobacterial enzyme by single site-directed mutagenesis.