Pulsating heat pipes (PHPs) are complex heat transfer devices whose thermal performance is governed by a strong thermohydrodynamic coupling. Recently, PHPs have attracted attention as novel electronic cooling devices. In this study, we used a self-rewetting fluid and obtained new experimental results for the improvement of the heat transport efficiency in PHPs. In contrast to the case of common liquids, the surface tension of self-rewetting fluids increases with temperature. Because of the increase in the surface tension at high temperatures, these fluids tend to flow toward the dry spot appearing on a heated surface, and thus, the boiling heat transfer is improved. We constructed PHPs from multiport extruded aluminum tubes with square channels. The PHPs consisted of a heating section, an adiabatic section, and a condensation section with a heat sink. We investigated the effect of the type of working fluid and the fluid fill ratio on the device performance. The working fluids employed were a self-rewetting fluid, water, and ethanol. The thermophysical properties of the working fluid affected the device performance, which also depended strongly on the boundary conditions employed during the PHP operation. In particular, the use of a self-rewetting fluid in the PHPs helped enhance the heat transport efficiency to a considerable extent.