A systematic study was carried out to assess property changes in CaCu3Ti4O12 thin films upon annealing in air in order to evaluate the impact of oxygen vacancies on their colossal dielectric constant behavior. Highly preferentially oriented thin-film samples were deposited by pulsed laser deposition method at 720 °C in 200 mTorr oxygen. The as-deposited thin films show the typical feature of colossal dielectric constant. Annealing experiments were conducted at 480, 580, 620, and 680 °C with intermediate electrical measurements. The evolutions of the electrical properties (such as, dielectric constant, loss, resistance, etc.), defect concentration and activation energy, were observed with the increase in the annealing temperature. Capacitance-temperature results exhibited the double-plateau feature in the measuring temperature range, revealing two defect levels in the sample. The activation energy and concentration of these two defects were estimated at each annealed state. The sensitivity of these defects to oxygen atmosphere confirmed our previous conclusion about oxygen vacancies as the chemical origin of the colossal dielectric constant phenomenon in CaCu3Ti4O12. Numerical simulation to the capacitance response of a Schottky junction with two defects supports the experimental results. Annealing at 680 °C changed the sample from the colossal dielectric state into a normal dielectric state with a negative temperature coefficient of dielectric constant. In such a fully annealed state, the intrinsic dielectric properties of CaCu3Ti4O12 were observed. The abnormal increase in dielectric constant at low temperature may be attributed to the magnetodielectric coupling caused by magnetic ordering.