A low-temperature scanning tunneling microscope (STM) and ab initio calculations were used to study the electric field effect on the vibration of single CO. molecules in an STM junction at 13 K. The vibrational energy of CO molecules adsorbed on silver atoms, measured by STM-based inelastic electron tunneling spectroscopy, depends on the direction of the electric field applied between the STM tip and the silver species. This characteristic can be explained by the charge separation model. The electric field modifies the binding characteristics of CO on silver as a result of a change in the charged states of the species, which leads to an increase (or a decrease) of the energies of the hindered rotation and the CO stretch on silver.