The effect of thiolate ligands was explored on the catalysis of CeO2 rod supported Au-25(SR)(18) (SR = -SCH2CH2Ph) by using CO oxidation as a probe reaction. Reaction kinetic tests, in situ IR and X-ray absorption spectroscopy, and density functional theory (DFT) were employed to understand how the thiolate ligands affect the nature of active sites, activation of CO and O-2, and reaction mechanism and kinetics. The intact Au-25(SR)(18) on the CeO2 rod is found not able to adsorb CO. Only when the thiolate ligands are partially removed, starting from the interface between Au-25(SR)(18) and CeO2 at temperatures of 423 K and above, can the adsorption of CO be observed by IR. DFT calculations suggest that CO adsorbs favorably on the exposed gold atoms. Accordingly, the CO oxidation light-off temperature shifts to lower temperature. Several types of Au sites are probed by IR of CO adsorption during the ligand removal process. The cationic Au sites (charged between 0 and +1) are found to play the major role for low-temperature CO oxidation. Similar activation energies and reaction rates are found for CO oxidation on differently treated Au-25(SR)(18)/CeO2 rod catalysts, suggesting a simple site-blocking effect of the thiolate ligands in Au nanocluster catalysis. Isotopic labeling experiments clearly indicate that CO oxidation on the Au-25(SR)(18)/CeO2 rod catalyst proceeds predominantly via the redox mechanism where CeO2 activates O-2 while CO is activated on the dethiolated gold sites. These results point to a double-edged sword role played by the thiolate ligands on Au-25 nanoclusters for CO oxidation.