The reversible and cooperative activation process, which includes electron transfer from surrounding redox mediators, the reversible valence change of cofactors and macroscopic functional/structural change, is one of the most important characteristics of biological enzymes, and has frequently been used in the design of homogeneous catalysts. However, there are virtually no reports on industrially important heterogeneous catalysts with these enzyme-like characteristics. Here, we report on the design and synthesis of highly active TiO2 photocatalysts incorporating site-specific single copper atoms (Cu/TiO2) that exhibit a reversible and cooperative photoactivation process. Our atomic-level design and synthetic strategy provide a platform that facilitates valence control of co-catalyst copper atoms, reversible modulation of the macroscopic optoelectronic properties of TiO2 and enhancement of photocatalytic hydrogen generation activity, extending the boundaries of conventional heterogeneous catalysts.Reversible and cooperative activation processes are important characteristics of biological enzymes and can be used in designing catalysts. Highly active TiO2 photocatalysts incorporated with site-specific single copper atoms are now shown to exhibit such a photoactivation process.