Exploring the active sites and reaction mechanisms of CO oxidation on metal oxides is of great significance in the field of heterogeneous catalysis. NiO has been attracting increasing attention in this field due to its high performance and low cost. Nevertheless, the active sites and reaction mechanism of NiO still remain controversial due to the complexity of the experiments involved, the limitations of characterization techniques, and the difficulty in searching for the global reaction pathway in theory. In this work, terraced and stepped NiO(100) surfaces, with and without oxygen vacancies, were established based on the Wulff construction, and the active sites and reaction mechanism were revealed at the atomic level using a novel global pathway searching method. Theoretical results indicate that the coordination-unsaturated Ni ions are the active sites for CO oxidation; O₂ interacts with the low-coordinated Ni ions to form reactive oxygen species; then they react with CO to form CO₂; oxygen species on stepped NiO(100) have a low barrier and sustain a catalytic cycle. The present work reveals that the best direction for the design and development of NiO-based catalysts with high performance is to prepare NiO catalysts with more defects and low-coordinated Ni ions. We anticipate that the approach adopted in this work can be applied to a wide range of heterogeneous catalysts for exploring the active sites and mechanisms.