[en] Highlights: • The spatial distribution of oxygen vacancies in heterostructured photoanodes were investigated. • Significantly enhanced photoelectrochemical performance is achieved by defect modulation. • Photoactivity of Bi₂WO₆ was boosted by suppressing intrinsic oxygen vacancies in W-O-W layers. • Oxygen vacancies around interface of Bi₂WO₆ and in TiO₂ side is beneficial for charge separation. Oxygen deficiency control has become an on-looming strategy for improving the catalytic ability of semiconductors, while the impact of defect distribution on the separation of charge carriers is still an open question. Herein, TiO₂/Bi₂WO₆ heterostructures are used as a typical model to demonstrate the hypothesis of boosting photoactivity of photoanodes through modulating the spatial distribution of oxygen vacancies. Compared to pristine TiO₂, significantly improved photoelectrochemical performance is achieved through suppressing intrinsic defects in Bi₂WO₆ and tuning the formation sites of interfacial oxygen vacancies. Both experimental and theoretical investigations demonstrate that the distribution of interfacial oxygen vacancies around interface of Bi₂WO₆ and in the TiO₂ side is beneficial for the efficient extraction of photogenerated electrons toward counter electrodes. This research shed atomic-level insight into the interfacial modulation of defect distribution. Therefore, it provides a new principle to develop efficient heterostructures for photoelectrochemical and photocatalytic applications.