The metal/oxide interface has been extensively studied due to its importance for heterogeneous catalysis. However, the exact role of interfacial atomic structures in governing catalytic processes still remains elusive. Herein, we demonstrate how the manipulation of atomic structures at the Au/TiO2 interface significantly alters the interfacial electron distribution and prompts O-2 activation. It is discovered that at the defect-free Au/TiO2 interface electrons transfer from Ti3+ species into Au nanoparticles (NPs) and further migrate into adsorbed perimeter O-2 molecules (i.e., in the form of Au-O-O-Ti), facilitating O-2 activation and leading to a ca. 34 times higher CO oxidation activity than that on the oxygen vacancy (V-o)-rich Au/TiO2 interface, at which electrons from Ti3+ species are trapped by interfacial V-o on TiO2 and hardly interact with perimeter O-2 molecules. We further reveal that the calcination releases those trapped electrons from interfacial Vo to facilitate O-2 activation. Collectively, our results establish an atomic-level description of the underlying mechanism regulating metal/oxide interfaces for the optimization of heterogeneous catalysis.