[Fe]-hydrogenase reversibly catalyzes the transfer of a hydride ion from H2 to methenyl-tetrahydromethanopterin (methenyl-H4MPT+) to form methylene-H4MPT. Its iron-guanylylpyridinol (FeGP) cofactor plays a key role in H2 activation. Here, we show that [Fe]-hydrogenase becomes O2-sensitive under turnover conditions in the presence of the reducing-substrates, methylene-H4MPT or methenyl-H4MPT+/H2. Only then, H2O2 is generated and decompose the FeGP cofactor as demonstrated by spectroscopic analyses and the crystal structure of the inactivated enzyme. O2 reduction to H2O2 requires a reductant, which can be a catalytic intermediate transiently formed during the [Fe]-hydrogenase reaction. The most probable candidate is an iron-hydride species; its presence has already been predicted by theoretical studies of the catalytic reaction. Our finding supported the prediction because the same type of reduction reaction is described for ruthenium hydride complexes that hydrogenate polar compounds. In addition, I will talk about the catalytic mechanism of [Fe]-hydrogenase.