As an environmentally friendly natural fluid with excellent thermodynamic and transport properties, carbon dioxide is an effective alternative refrigerant. This study presents an experimental investigation of an R744-based two-phase thermosyphon loop (LTS) used to cool a data center. With a heat transfer rate change of 1–4 kW, the thermal resistance and driving temperature difference of the LTS are examined. The total thermal resistance decreases with increasing heat transfer rate due to the reinforced flow boiling heat transfer of R744. The R744 LTS can work under a small driving temperature difference of 5 °C. According to a contrast experiment, the R744 LTS has superior heat transfer performance, with a driving temperature difference that is 2 °C smaller than that of an R22 LTS when the heat transfer rate is 2 kW. Besides, a theoretical model is also used to analyze the performance of the LTS. The calculated results show that the air-side thermal resistance accounts for the majority (68–82%) of the total thermal resistance.