Cu clusters supported on CeO2 nanorods (NRs) were found to exhibit significantly enhanced activity and thermal/hydrothermal durability for low-temperature CO oxidation when the catalyst was calcined at 800 °C in air prior to the catalytic measurements. The effect of calcination temperature and Cu loading on Cu-CeO2 catalysts was studied by the combination of powder X-ray diffraction (PXRD), Raman spectroscopy, H2-temperature program reduction (H2-TPR), ambient-pressure X-ray photoelectron spectroscopy (APXPS), synchrotron radiation photoelectron spectroscopy (SRPES), and X-ray absorption spectroscopy (XAS). Cu atoms were found to incorporate into the ceria lattice in air at below 500 °C, leading to the formation of a bulk CuyCe1–yO2–x solid solution. As the calcination temperature was raised to 800 °C, the solubility of Cu ions in bulk ceria was reduced sharply. Surface segregation of Cu atoms led to the formation of CuOx and a surface Cu-doped ceria thin layer, resulting in a much enhanced activity for CO oxidation. Moreover, the activities of calcined Cu-CeO2 catalysts did not increase with the Cu loading but exhibited an optimal activity at ∼2 wt % of Cu loading, where segregated CuOx species gave an average size in the sub-nanometer (sub-nm) range. Larger CuOx nanoparticles (NPs) on the Cu-doped ceria thin layer exhibited a lower activity toward CO oxidation. We believe the enhanced catalytic properties of the thermally aged Cu-CeO2 catalysts are attributed to the formation of an interface between sub-nm CuOx and the Cu-doped ceria thin layer. Our study thus sheds light on the nature of most active sites on Cu/CeO2 catalysts and facilitates the rational design of ceria-supported metal catalysts for oxidation reactions.