Understanding the reaction mechanism and the nature of the reactive species of heterogeneous catalysts under reaction conditions is the first step in the design of more consistent, reliable, and practical catalysts. We used density functional theory (DFT) calculations to study the mechanism of CO oxidation catalyzed by CeO2-supported Au nanoparticles (NPs) under reaction conditions by considering the sequential CO adsorption onto and CO saturation of Au NPs. We found that the Au9NPs supported by CeO2(100) and CeO2(111) bind as many as eight or four CO molecules, respectively. The last-bound CO molecule opens the fast CO oxidation pathway. The CO oxidation pathways constructed on both systems show that the reaction occurs at the Au–CeO2interface via the Mars–van Krevelen mechanism. We found that the most important O–C–O-type intermediate was spontaneously formed at the Au–CeO2(100) interface upon the sequential binding of CO molecules onto the Au NPs. The reaction pathway therefore becomes relatively simpler than the CO oxidation pathways constructed with a first-bound single CO molecule. Although the O–C–O formation at the Au–CeO2(111) interface requires overcoming an activation energy barrier, the rate of CO oxidation shows that the Au/CeO2(111) was also highly reactive even at room temperature. Our findings show that the surface of Au NPs supported by CeO2will be saturated with CO under CO oxidation conditions and that subsequent CO oxidation occurs at the Au–CeO2interface. Our results suggest that the interaction between the catalysts and the reacting molecules should be more intensively studied to understand the catalytic performance of supported NP catalysts under reaction conditions.