Although defects play an important role in the photocatalytic activity of TiO2, the mechanism of the photocatalytic activity related to different defects remains disputable. Moreover, the reported methods to introduce defects raise the preparation cost. In this work, different types of defects including O-vacancy cluster, surface O-vacancy, and bulk O-vacancy defects are in situ introduced in TiO2 by controlling the crystallization temperature. The medium-degree crystallinity TiO2 sample mainly containing surface O-vacancies exhibits the best NO removal activity. The systematic study of photocatalytic mechanism demonstrates that the surface O-vacancies significantly promote the adsorption of H2O molecules and improve charge transfer to the adsorbed H2O forming center dot OH, thus dramatically enhancing the photocatalytic NO removal activity. On the contrary, bulk O-vacancies neither help the adsorption of H2O molecules, nor improve the charge transfer to H2O.