A Fourier transform infrared (FTIR), UVis, and high-resolution transmission electron microscopy (HRTEM) study of fresh and used Au/ZrO2 catalyst is reported. FTIR data show that on a fresh sample CO is adsorbed on regular gold metallic sites, while on the used sample the adsorption sites are bidimensional gold clusters made positive by interaction with the support. NIR-UV-vis reflectance spectra show that the gold plasmonic peak appears broader and the ZrO2 absorption edge weaker on the used sample with respect to the fresh one. HRTEM indicates the disappearance of the gold metallic particles in the used catalysts. Moreover, a strong reduction in the CO oxidation activity has been evidenced on the used sample and ascribed to the positivization of gold and/or to the decrease of the number of highly uncoordinated gold sites, consistently with the spreading of gold at the support surface.