The phasing out of hydrochlorofluorocarbon and chlorofluorocarbon fluids and further environmental problems arising from new, synthetic working fluids stimulate a continuously rising interest on natural candidates. The nontoxic and nonflammable CO2 impacts neither on ozone depletion nor on global warming if leaked to the atmosphere. The critical temperature of CO2 (31.1 °C) is almost ambient and, therefore, it undergoes a trans-critical refrigeration cycle. In cooling mode operation, a trans-critical CO2 system, as compared to conventional air-conditioners, has a lower performance which, contrary to conventional systems, strongly depends on the refrigerant charge. In this paper, the performance of the trans-critical system was evaluated experimentally under different refrigerant charge amounts. The influence of charge on coefficient of performance (COP), cooling capacity, compression ratio, suction line superheat was analyzed in detail. The experimental results indicate that the COP of the trans-critical cycle attains a maximum at optimal refrigerant charge. By varying the charge the cooling capacity attains a maximum, as well, that corresponds to the optimal charge. In order to understand the effect of refrigerant charge on the performance of each device of the plant, an exergetic analysis based on the experimental data was carried out. The analysis shows that the exergy flow destroyed in the compressor is one of the major causes of overall exergy destruction. At the optimal refrigerant charge, the compression losses attain a minimum.