MoO3-x thin films have been prepared by pulsed laser deposition (PLD). The oxygen-vacancies could not only greatly increase the interlayer spacing and the electrical conductivity of MoO3, but also significantly enhance the electrochemical activity, which leads faster charge storage kinetics. The oxygen vacancies have been characterized using a series of techniques: X-ray absorption fine structure, X-ray absorption near edge structure. Through a combination of density functional theory and experiments, the role of the oxygen vacancy sites in the redox reaction activity of MoO3-x is identified. The MoO3-x thin film exhibited good reversibility. And the reversible discharge capacity of above 520 mA h g-1 was achieved after 100 cycles. In further data analysis, the differences between the compositions are investigated, the concentration of oxygen vacancies is estimated, and the possible effects on the cation defect chemistry and phase formation of MoO3-x are discussed. The reaction mechanism of MoO3-x thin-film electrode has been investigated by scanning electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. These results confirmed that incorporating oxygen vacancies into transition metal oxidesiis is an effective strategy for increasing the material's electrochemical reactivity.