Free cooling based on loop thermosyphon is an ideal method for energy-saving of data centers. In order to promote its future application, it is necessary to investigate the performance of utilizing environment friendly working fluids. In this paper, an experiment based on enthalpy difference method and thermal imaging is conducted on a CO2 loop thermosyphon with microchannel parallel-flow evaporator and condenser. The results show that the effect of filling ratio on CO2 loop thermosyphon is more significant and the optimal filling ratio is higher, compared with R22 loop thermosyphon. Thermal image of the evaporator shows that superheated region shrinks and then disappears with the increase of filling ratio. Moreover, to analyze the relationship between the phenomenon and the internal flow state, a distributed-parameter model is built and validated. The simulation results show that 150% is the optimal filling ratio for no superheating or subcooling occurs along the loop. The effects of height difference and pipe length are more significant for low filling ratio. Superheating is easier to occur at the evaporator outlet when height difference is smaller or pipe length is larger, and subcooling is easier to occur at the condenser outlet when height difference or pipe length is smaller.