Co-catalysts play an important role in photocatalytic water splitting. The co-catalyst is generally deposited in the form of nanoparticles on the catalyst surface, and is believed to provide water oxidation and reduction sites. However, the minimum size of a co-catalyst that can function as a reaction site and the detailed local environment of the photocatalytic reaction centers are not yet fully understood. Here, we show that even isolated single-atom Rh dopants in two-dimensional titanium oxide crystals can effectively act as co-catalysts for the water-splitting reaction. At an optimal doping concentration, the hydrogen production rate is increased substantially in comparison to that found with the undoped crystals. We also present first-principles simulations based on density functional theory to provide insights into the atomic-scale mechanism by which the isolated Rh dopants induce changes to the dissociation reaction energy landscape. These results provide new insights for better understanding the role of the co-catalyst in the photocatalytic reaction.