This study investigated the impact of high-voltage electric field (HVEF) thawing technology on the thawing rate, water retention characteristics, microstructure, and nutritional composition of thawed beef. Compared with the control group, in which thawing occurred under natural conditions, the experimental group, in which beef was thawed under HVEF (12 kV, 16 kV, 20 kV, 24 kV, 28 kV), showed a significantly shorter thawing time, and the higher the voltage was, the faster the thawing rate. The total loss rate of thawed beef reached its minimum value of (54.2 ± 0.62) % at 28 kV, and the water retention of the experimental group was significantly better than that of the control group (p < 0.05). Therefore, it can be concluded that HVEF thawing enhances the water retention ability of beef. In a color comparison, it was evident that the color of the beef thawed by HVEF was significantly better than that of the control group. The results of scanning electron microscopy (SEM) indicated that thawing beef by HVEF can reduce the damage to the myofibril structure. Low-field nuclear magnetic resonance (LF-NMR) showed that beef thawed by HVEF had a significantly increased bound water content. According to the determination of malondialdehyde content, beef thawed by HVEF had a reduced degree of lipid oxidation; the content at 16 kV was 2.4 mg/kg, and the degree of lipid oxidation was the lowest. Fourier transform infrared (FTIR) spectroscopy analysis revealed that the absorption peak positions of the beef samples did not show significant changes under different conditions. However, the absorption peak intensity in the experimental group was generally higher than that in the control group. Examination of the protein secondary structure via infrared spectroscopy revealed that, compared with the control group, HVEF thawing transformed the proteins from an ordered structure to a disordered structure. The increase in disordered structure reduced the fiber gap of the sample and improved the water retention of the beef. The above experimental results indicate that HVEF thawing can improve the water-holding capacity of the sample and reduce the thawing damage to the quality of the sample.