We report our experimental study of the adsorption and diffusion of CO on clean TiO2(110)-1 x 1 at an atom-resolved scale with in situ scanning tunneling microscopy at 80 K, combined with density functional theory calculations. Our results reveal that the CO adsorption sites on the TiO2(110)-1 x 1 surface are preferentially located at nextnearest-neighbor five-coordinate Ti atoms close to a bridge-bonded oxygen vacancy (BBOv), in contrast to the idea that the BBOv itself acts as the adsorption site for CO. The equilibrium distribution of CO adsorption on the TiO2(110)-1 x 1 surface can be well-understood according to the calculated adsorption energies of different sites.