Abstract: Prestressed reinforced concrete structures are still the commonly used structural form for containment buildings in large pressurized water reactor nuclear power plants. In addition to meeting basic functional requirements, the structures of nuclear power plants may also face various impact events, such as impacts from tornado-borne projectiles, accidental crashes of small airplanes, and the impacts of commercial airliner collisions. Conducting research on the penetration resistance of prestressed reinforced concrete structures can provide important references for assessing and analyzing the structural state in relevant accident scenarios. At present, the basic experimental research on the penetration resistance of prestressed reinforced concrete structures is lacking, and the research on the enhancement effect of the existence of prestressing on the structural penetration resistance is insufficient. Therefore, the experimental research on the penetration resistance of prestressed reinforced concrete structures was conducted and a numerical simulation method through experimental calibration was established. On this basis, the enhancement effect of prestressing on the penetration resistance of the structure was further explored. The experimental results indicate that prestressing has a minimal impact on the depth of the cratering on the front face of the concrete target, but the application of initial prestressing increases the extent of the cratering on the front face. Further numerical simulations were conducted to reproduce the entire experimental process. The numerical simulation results are very consistent with the experimental results, validating the effectiveness of the constitutive model, parameter selection, and finite element analysis method used. Finally, based on the numerical simulation method, the anti-penetration ability of prestressed reinforced concrete target plate under different penetration speeds was calculated and analyzed. The overall analysis results indicate that prestressing has a minor effect on the penetration resistance of the target plate. The increased confining pressure due to prestressing increases the resistance encountered by the projectile passing through the target plate, resulting in a reduced residual velocity of the projectile compared to that when it penetrates a normal target plate.