Zeolites have well-defined pores of molecular dimensions to stabilize the molecularly dispersed reactive oxygen species (ROS) responsible for actual key intermediates in a variety of desirable catalytic transformation processes. Defining the local environment to create such an important class of ROS is undoubtedly important for designing more efficient zeolite-based catalysts; however, it remains challenging due to the obscure distribution of the framework Al atoms. In the present study, we defined the local environment of MFI zeolite to create a novel O 2 activation property of the cobalt ion. We found that the Co III −peroxo species is formed through two-electron reduction of O 2 at room temperature by an extra lattice Co I species that corresponds to an abnormal oxidation state of Co. This is the first example of the experimental detection of the O 2 -derived mononuclear metal−peroxo species in zeolites. The change in the skeletal vibrational mode associated with the Al site, ν Al−O−Si , upon the O 2 activation process was experimentally observed. It provides the information about the position of the framework Al atom. By applying B3LYP-D3 density functional theory (DFT) calculations, we demonstrated that the positioning of the framework Al atom at an interface between straight and sinusoidal channels of MFI zeolite is essential for the formation of the Co III −peroxo species. The experimentally calibrated DFT model features the S = 1 square planar Co III −peroxo species bound to the interface region by two anionic lattice oxygen atoms. The Co III −peroxo species is stabilized only by weak donor O ligands, never previously identified. This unique state is demonstrated by combined experimental and computational analyses of the vibronic progression found for the first time in this system, which provides detailed information about the site geometry and electronic structure. The findings of the present study assist us to understand how the zeolite lattice creates the abnormal O 2 activation property of metal ions.