Plasticizers and the water migration of edible protein films during storage can result in changes in film properties, while specific changing processes need to be further explored. In this study, glycerol-plasticized soy protein isolate (SPI) films were stored at 25 °C, 4 °C, and −18 °C for 6 weeks (relative humidity (RH), 40–50%). The glycerol migration was monitored by the glycerol migration rate and differential scanning calorimetry (DSC). Water content, low-field nuclear magnetic resonance (LF-NMR), and thermogravimetric analysis (TGA) were used to analyze the water state. The results showed that significant pores and cracks were observed after storage at 25 °C. The proportion of bound water gradually increased, and the glycerol migration rate also reached 1.3% and 0.7% at 25 °C and 4 °C, respectively. The results proved that increasing the storage temperature accelerated the loss of water and glycerol, and decreased the mechanical properties of the SPI film.