Understanding the mechanism by which oxygen adsorption influences the separation behavior of charge carriers is important in photocatalytic removal of air pollutants. In this study, we performed steady-state surface photovoltage and surface photocurrent spectroscopy combined with an atmosphere control system to determine the effect of oxygen on the charge separation behavior at the surface of anatase TiO2 nanoparticles at ambient temperature. Results showed that photogenerated electrons were movable in N2 atmosphere but were localized in O2 atmosphere. O2 obviously enhanced the stabilization of the localized photogenerated electrons when the surface defects of TiO2 were fully occupied by adsorbed O2. Moreover, O2 adsorption increased the energy demand for exciting electrons from the valence band to localized surface defect states and reduced the density of band tail states. These findings suggest us that the effect of gaseous species on the mobility and stability of charge carriers should be considered to understand the photocatalytic degradation of air pollutants.