Ammonium vanadate (NH4V4O10) is regarded as a promising cathode material for aqueous zinc-ion batteries, given its considerable theoretical capacity and tunable interlayer spacing. However, due to its inherent low electrical conductivity and the excessive presence of ammonium ions between layers, NH4V4O10 exhibits unsatisfactory electrochemical performance. In this study, it is proposed that the electrochemical performance of NH4V4O10 can be significantly enhanced by removing part of the ammonium cation and increasing the vanadium vacancy. The decrease of ammonium further increases the layer spacing, reduces the irreversible deamination and accelerates the diffusion of Zn2+. Density functional theory (DFT) calculations reveal that increasing vanadium vacancies significantly diminishes the strong electrostatic interactions between the V-O layers and Zn2+, enhances the material’s electrical conductivity, and stabilizes the crystal structure during zinc ion (de)intercalation, thus obtaining excellent electrochemical properties. As a result, the Vd-NVO cathode demonstrates a high reversible capacity of 371 mAh g−1, excellent rate performance, and remarkable cyclic stability, maintaining a reversible capacity of 131 mAh g−1 even after 1000 cycles at 5 A g−1.