Photoactivatable (PA) fluorescent proteins are a new class of fluorescent proteins in which the intensity of fluorescence is dramatically enhanced through photoinduced decarboxylation process. In the present study, the reaction mechanism of the photoinduced decarboxylation in PA-GFP was investigated by the ONIOM(QM:MM) method. The decarboxylation process starts from the first excited state (IntraCT state) and then proceeds along an InterCT state after the first crossing (or an approximate transition state). Relative to an equilibrium structure in So, a barrier of similar to 94 kcal/mol to reach this approximate transition state is the rate-determining step for the entire decarboxylation process. The InterCT state becomes the open-shell ground state in the product, after the subsequent crossing with a closed-shell state that holds an extra electron on the dissociated CO2. The present study elucidated for the first time the mechanism of the photoinduced decarboxylation of PA-GFP and supports the widely accepted Kolbe pathway, which could be a common mechanism for the irreversible photoinduced decarboxylation in different fluorescent proteins.