The regulation of interfacial defects of nanocrystals aligned orderly in a superstructured photocatalyst is an effective approach to improve the photocatalytic performance. However, the synthesis of ordered self-assemblies with abundant interfacial defects and reactive sites is hard to achieve, and applications are limited due to the unclear physicochemical properties, which result from the unique mesocrystalline microstructures. It is reported herein that the photocatalytic properties depend on the interfacial defects of intergrains in anatase TiO mesocrystals (TMCs). Research reveals that the photocatalytic activity largely depends on defects, such as lattice distortion and oxygen vacancies, which are located at highly aligned interfaces of intergrains within TMCs. Moreover, the mesocrystalline TiO photocatalysts exhibit higher photocatalytic performance in organic degradation and hydrogen evolution, compared with single crystals and polycrystals; this can be ascribed to an appropriate amount of interfacial defects at the intergrains and improved carrier separation efficiency through the highly oriented interfaces. In addition, the photocatalytic performance of the TMCs could be further improved through regulation of defects by undergoing an annealing process under a redox atmosphere. This work can provide an avenue to defect engineering for improved photocatalytic performance.