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The use of carbon dioxide (CO2) as refrigerant is driven by the need to phase down the traditional synthetic refrigerant so as to mitigate the warming climate. In this study, the thermal performance of a two-stage compression transcritical CO2 refrigeration system with R290 mechanical subcooling unit is conducted. The goal is to obtain the maximum coefficient of performance (COP) of five different mechanical subcooling systems under the optimal subcooling temperature and compressor discharge pressure. The two-throttling and two-stage compression high-pressure mechanical subcooling system is found to have a higher COP. When the evaporation temperature is −30 °C, the COP of the two-throttling and two-stage compression high-pressure mechanical subcooling system is improved by 76.74%. The COP of the two-throttling and two-stage compression high-pressure mechanical subcooling system is 1.52 at an ambient temperature of 40 °C, which is 21.87% higher than that of the two-throttling and two-stage compression low-pressure mechanical subcooling system. The power consumption ratio of one-throttling and two-stage compression low-pressure mechanical subcooling system is significantly higher than that of other systems. From a comprehensive analysis of the proposed four systems, the two-throttling and two-stage compression high-pressure mechanical subcooling system has the best performance over all other systems.