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 mu-oxo (O5) and the additional oxygenic ligand (Ox) of the manganese-calcium oxide cofactor. However, the subsequent process through which molecular dioxygen is formed and released remains elusive. In this report, quantum chemical calculations reveal that the O-2 release 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-O-2 where the Mn-Ox bond is cleaved, and after a twist of the quasi-O-2 unit, the free O-2 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 intricate mechanism involved in an important step of photosynthetic oxygen evolution for a deeper understanding of nature's water oxidation catalysis.