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Nafion membranes are now the most widely used membranes for long-life vanadium flow batteries (VFBs) because of their extremely high chemical stability. Today, the type of Nafion membrane that should be selected and how to pretreat these Nafion membranes have become critical issues, which directly affects the performance and cost of VFBs. In this work, we chose the Nafion 115 membrane to investigate the effect of the pretreatment process (as received, wet, boiled, and boiled and dried) on the performance of VFBs. The relationship between the nanostructure and transport properties of Nafion 115 membranes is elucidated by wide-angle X-ray diffraction and small-angle X-ray scattering techniques. The self-discharge process, battery efficiencies, electrolyte utilization, and long-term cycling stability of VFBs with differently pretreated Nafion membranes are presented comprehensively. An online monitoring system is used to monitor the electrolyte volume that varies during the long-term charge-discharge test of VFBs. The capacity fading mechanism and electrolyte imbalance of VFBs with these Nafion 115 membranes are also discussed in detail. The optimal pretreatment processes for the benchmark membrane and practical application are synthetically selected.