Stabilizing the oxygen vacancies (Ovs) in ZnO at high temperatures is crucial for maintaining the high thermoelectric (TE) performance caused by Ovs. In this study, based on the complex interaction between different kinds of lattice defects, high-density stacking faults (SFs) were fabricated in ZnO with Ovs to effectively stabilize Ovs. The Ovs combined with SFs to significantly improve the electrical conductivity (σ) to 4649 S m−1 at room temperature, leading to an increased power factor (PF) of 280.4 μW m−1·K−2 at 500 °C. Meanwhile, the thermal conductivity was reduced to 3.12 W m−1·K−1 at 500 °C. Notably, the σ and PF were not weakened after five consecutive cycle tests up to 500 °C, suggesting the stabilization of Ovs by SFs at high temperatures. Consequently, the optimized ZT value reached 0.07 at 500 °C, an 18-fold enhancement over the initial sample. The study provides an effective strategy to achieve a simultaneous enhancement in TE performance and stability by modulating different kinds of microstructures.