Symmetry breaking induced by interfaces may produce abundant abnormal physical phenomena. The novel phenomena generated by these interfaces inspire people to explore the physics of oxide interface engineering. For LaCoO 3−x materials, it is undoubtedly critical to understand the structure variations of thin films caused by different interface conditions for the successful application of thin films in electronic devices such as solid oxide fuel cells, oxygen separation membranes, gas sensors, rechargeable batteries, memristors, etc. Oxygen vacancy configurations and concentration are coupled with the magnetic, electronic, and transport properties of perovskite oxides, and manipulating the physical properties by tuning the vacancy structures of thin films is crucial for applications of these functional devices. Here, we report a direct atomic-scale observation of the variation of the oxygen vacancy structure in strained LaCoO 3−x films under different boundary conditions. Using aberrationcorrected transmission electron microscopy, we observe that in an 8 nm LaCoO 3−x film grown on the SrTiO 3 substrate, the ordered oxygen vacancies stack layer by layer along the in-plane direction with a period of 3 unit cells (UCs) and appear in a staggered-arrangement way. When a 20 nm BiFeO 3 film is grown on top of the LaCoO 3−x film, the oxygen vacancy concentration in the LaCoO 3−x film reduces and becomes very low. Different from that in free upper surfaces, the oxygen vacancy concentration is too low to be ordered. When a 20 nm PbTiO 3 film is grown on the LaCoO 3−x film, the oxygen vacancy concentration in the LaCoO 3−x film is very high, and different from that of the free upper surface, the ordered oxygen vacancies stack layer by layer along the out-of-plane direction with a period of 2 UCs, and the oxygen vacancies stack in a nonstaggered-arrangement way. Further reducing the film thickness, when a 6 UC PbTiO 3 film is grown on the 8 UC LaCoO 3−x film, the oxygen vacancy concentration in the LaCoO 3−x film is lower than that of 20 nm PTO/8 nm LCO/STO, and the ordered oxygen vacancies still stack layered along the out-of-plane direction with a period of 2 UCs, but the oxygen vacancies stack in a staggered-arrangement way. First-principles calculations decipher the atomic-scale mechanism of oxygen vacancy distribution. These results suggest that oxygen vacancy distribution is heavily associated with interface configurations. These studies are of great significance for understanding the behavior of oxygen vacancy and guiding the design of different interfaces to achieve the appropriate structure and performance for functional electronic devices.