Owing to the high energy density, high security and low price, rechargeable magnesium batteries (RMBs) are promising candidate for the next generation of high-performance batteries. However, the development of cathode materials for RMBs is hindered by the intensive polarization of Mg2+, which tends to destroy the stability of crystal structure and results in the degradation of batteries. Pre-intercalating the different alkali ions in crystal structure is an effective strategy to improve the layered structure stability and electrochemical performance of materials. Herein, the alkali ions (Li+, Na+, K+) pre-intercalation is presented to improve the structure stability of layered vanadium oxide (A-V3O8) for Mg2+ storage. From the result, the cycling performance of cathode is promoted with the pre-intercalation radius increase. To explain the optimizing principle, we use density functional theory (DFT) calculation to simulate the interaction effect between pre-intercalated cation and layered structure. Among intercalation compounds A-V3O8 (A = Li, Na, K), the electrochemical performance of Na+ pre-intercalated materials (NaV3O8) is better than the most of cathode materials for RMBs. Besides, the reaction mechanism of NaV3O8 is demonstrated. This work confirms that the pre-intercalation of appropriate alkali cation is an efficient strategy to improve the electrochemical performance of layered cathode materials for RMBs.