<p>Developing highly active and stable electrocatalysts based on the lattice oxygen mechanism (LOM) for the oxygen evolution reaction (OER) represents a significant challenge in water splitting. Herein, we successfully introduce oxygen vacancies (O_v) into high-entropy MnFeCoNiCu layered double hydroxides (HE-LDHs) via a solution chemical reduction method utilizing a defect engineering strategy. By precisely tuning the concentration of oxygen vacancies, we effectively activate the lattice oxygen within the HE-LDHs. The optimized O_v-rich high-entropy LDHs (O_v-HE-LDHs) exhibit excellent OER catalytic performance, achieving a current density of 10 mA cm⁻² with a remarkably low overpotential of only 210 mV in 1.0 M KOH electrolyte, which is substantially superior to pristine HE-LDHs (315 mV) and commercial IrO₂ (330 mV). Furthermore, the catalyst demonstrates outstanding long-term stability, capable of stable operation for 500 h at a high current density of approximately 200 mA cm⁻². Advanced X-ray absorption fine structure analysis elucidates the lower metal valence states, indicating the existence of oxygen vacancies, while isotope labeling experiments and <i>in-situ</i> electrochemical Raman spectroscopy strongly confirm the successful activation of the LOM pathway. Density functional theory calculations further validate that the shift in the OER mechanism towards LOM and the resulting reduction in the reaction energy barrier are the fundamental reasons for the catalyst’s enhanced intrinsic activity. This work proposes a novel strategy for activating lattice oxygen in high-entropy LDHs through defect engineering, offering new insights and experimental guidance for the design and development of highly efficient and stable high-entropy OER electrocatalysts.</p>