The understanding of O–O bond formation is of great importance for revealing the mechanism of water oxidation in photosynthesis and for developing efficient catalysts for water oxidation in artificial photosynthesis. The chemical oxidation of the Ru^II₂(OH)(OH₂) core with the vicinal OH and OH₂ ligands was spectroscopically and theoretically investigated to provide a mechanistic insight into the O–O bond formation in the core. We demonstrate O–O bond formation at the low-valent Ru^III₂(OH) core with the vicinal OH ligands to form the Ru^II₂(μ-OOH) core with a μ-OOH bridge. The O–O bond formation is induced by deprotonation of one of the OH ligands of Ru^III₂(OH)₂ via intramolecular coupling of the OH and deprotonated O⁻ ligands, conjugated with two-electron transfer from two Ru^III centers to their ligands. The intersystem crossing between singlet and triple states of Ru^II₂(μ-OOH) is easily switched by exchange of H⁺ between the μ-OOH bridge and the auxiliary backbone ligand.