Natural photosynthesis plays a vital role in the supply of energy and oxygen necessary for the survival of biological organisms. The current leading proposal of the O-O bond formation in photosystem II suggests the coupling between the central μ-oxo (O5) and the additional oxygenic ligand (Ox) of the manganese-calcium oxide cofactor. However, the subsequent process through which molecular dioxygen is formed remains elusive. In this report, quantum chemical calculations reveal that the O2 process is initiated by the cleavage of the Mn-O5 bond, without a preliminary conformational change of the peroxide [O5-Ox]2- group. Subsequently, the [O5-Ox] moiety is converted from the superoxide to the weakly bound quasi-O2 where the Mn-Ox bond is cleaved, and after a twist of the quasi-O2 unit, the free O2 is ultimately released. Alternative pathways display significantly slower kinetics, due to the lower structural stabilities of the rate-limiting transition states. The cause of the difference is associated with the Jahn-Teller axial orientation and the local ring strain within the Mn cluster. These findings contribute to unravelling the complex mechanism in an important step of photosynthetic oxygen evolution for a deeper understanding of nature's water oxidation catalysis.