Oxygen vacancy defects play an important role in determining the properties of zinc oxide (ZnO) film. In this study, ZnO film prepared by a high-deposition-rate spatial ALD process was annealed in an oxygen-rich ambient. The variation of oxygen vacancy defects was analyzed. The results show that oxygen vacancy defects were suppressed by the annealing treatment, particularly at 500 °C–700 °C. As a result, the carrier concentration decreases with increasing annealing temperature, leading to the higher film resistivity. In addition, the oxygen atoms segregated to the ZnO/Si interface to form an interfacial oxide layer, leading to an oxygen-deficient ZnO film when annealed at 300 °C–400 °C and the decrease of oxygen atomic ratio when annealed at 500 °C–800 °C. The annealing treatment also induces higher crystallinity with larger crystallite size, leading to a higher mobility. The refractive index increases with annealing temperature at 300 °C–700 °C, possibly due to the structural relaxation, densification and crystallization of the film. However, the annealing treatment has little effect on the film transmittance due to the low optical loss.