The current study investigated the heat transfer performance, operation limits, and temperature distribution of a 3 m prototype model for a large-scale straight two-phase closed thermosyphon with deionized water designed for passive cooling of nuclear spent fuel pools with a filling ratio ranging from 20% to 100% and a heat source temperature ranging from 45 to 80 °C. After the accident at the Fukushima Daiichi nuclear power plant, the need for a reliable cooling system for these pools increased, with thermosyphons emerging as a promising solution for passive cooling. The experimental procedure implemented in this study yielded a comprehensive understanding of the operation and phenomena inside a thermosyphon, providing crucial data for the validation and enhancement of numerical models that simulate relevant phenomena within nuclear power plants, including passive residual heat removal with thermosyphons. The results indicated that the optimal heat transfer performance was achieved at a filling ratio of 30%, where the thermosyphon begins operation at a heat source temperature of 45 °C. Additionally, the temperature distribution along the thermosyphon confirmed the operation limits, including partial dryout at a 20% filling ratio and geyser boiling and flooding (entrainment) limits at 75% and 100% filling ratios, respectively.