Iron-substituted manganese superoxide dismutase (Fe(Mn)SOD) was produced using an in vivo preparation method. It’s an inactive enzyme in catalyzing superoxide radical dismutation owing to the mis-incorporation of Fe in the active site evolved to use Mn. To investigate the possible toxicity of human Fe(Mn)SOD proposed by Yamakura, we studied the properties of Fe(Mn)SOD upon H2O2 treatment and compared to that of FeSOD. It’s found that the responses to H2O2 treatment were different, including the changes of optical spectra, variations of active site coordination and secondary structures. Fe3+ reduction was not observed in Fe(Mn)SOD even H2O2 is believed to oxidize proteins via highly reactive intermediates including Fe and formed via Fe2+, which is true in FeSOD. What’s more, the activities of Fe(Mn)SOD and FeSOD were totally different in the ABTS assay or Amplex Red assay. These results indicated that the mechanism of peroxidase reaction of Fe(Mn)SOD is not identical to that of FeSOD.