Interlayer engineering is a promising strategy to modify the structure of layered vanadium‐based oxides with optimized ion‐diffusion capability, during which the role of interlayer crystal water in tuning the charge storage properties should be clarified. Herein, a series of hydrated V 2O 5· nH 2O xerogels with varying contents of interlayer water ( n) is obtained by differentiating the temperature parameter. Results show that the value n should be properly modified to the best value that is not too large or too small, that is, when n equals 0.26, the V 2O 5· nH 2O electrode exhibits optimized discharge capacity (456.5 mAh g −1 at 0.1 A g −1) and cycling stability with 94.3% retention after 2,000 cycles at 3 A g −1. Regulation of interlayer water content appears to weaken the electrostatic interaction between the VO framework and intercalated Zn 2+, which thus enhances the reversibility of the zincation and structural evolution during cycling. Density functional theory calculations suggest that the V 2O 5·0.26H 2O material with modulated electron structure can provide a favorable electrostatic environment for reversible Zn 2+ diffusion with a lower migration barrier. The findings of this work are expected to arouse more intensive research efforts into the role of structural water in tuning the energy storage performance in various electrode materials.