Activation of molecular O₂ is the most critical step in gold-catalyzed oxidation reactions; however, the underlying mechanisms of this process remain under debate. In this study, we propose an alternative O₂ activation pathway with the assistance of hydrogen-containing substrates using density functional theory. It is demonstrated that the co-adsorbed H-containing substrates (R–H) not only enhance the adsorption of O₂, but also transfer a hydrogen atom to the adjacent O₂, leading to O₂ activation by its transformation to a hydroperoxyl (OOH) radical species. The activation barriers of the H-transfer from 16 selected R–H compounds (H₂O, CH₃OH, NH₂CHCOOH, CH₃CH=CH₂, (CH₃)₂SiH₂, etc.) to the co-adsorbed O₂ are lower than 0.50 eV in most cases, indicating the feasibility of the activation of O₂ via OOH under mild conditions. The formed OOH oxidant, with an increased O–O bond length of ~1.45 Å, either participates directly in oxidation reactions through the end-on oxygen atom, or dissociates into atomic oxygen and hydroxyl (OH) by crossing a fairly low energy barrier of 0.24 eV. Using CO oxidation as a probe, we have found that OOH has superior activity than activated O₂ and atomic oxygen. This study reveals a new pathway for the activation of O₂, and may provide insight into the oxidation catalysis of nanosized gold.