Thermally stable Au single-atoms supported by monolayered CuO grown at Cu(110) have been successfully prepared. The charge transfer from the CuO support to single Au atoms is confirmed to play a key role in, tuning the activity for CO oxidation. Initially, the negatively charged Au single-atom is active for CO oxidation with its adjacent lattice O atom depleted to generate an O vacancy in the CuO monolayer. Afterward, the Au single-atom is neutralized, preventing further CO reaction. The produced O vacancy can be healed by exposure to O-2 at 400 K and accordingly the reaction activity is restored.