Pulsating heat pipes (PHPs) are widely used in the heat dissipation of electronic components, waste heat recovery, solar energy utilization, etc., relying on the pulsating flow of the work material in the pipe and the heat transfer by phase change, and they have the advantages of high heat-transfer efficiency, simple structure, and low cost. In this paper, an experimental method is used to adjust the length of local pipes in the PHP structure, so that the PHP forms a high- and low-staggered asymmetric structure, and to study the effects of different liquid charging rates and heat-source temperatures on the vibration, startup, and operation of the PHP in the asymmetric structure. We found the following: it is difficult to start up and operate the workpiece at 10%, 68%, and 80% liquid charging rates; the effect of the oscillating impact is worse; the temperature difference between the evaporation section of the pulsating heat pipe and condensation section is larger; and the temperature difference between the evaporation section and condensation section is larger. The temperature difference between the evaporation section and condensation section of the pulsating heat pipe is large, the temperature difference is between 10~25 °C, and it is difficult to achieve a small temperature difference in heat transfer. When the liquid charging rate is 30% and 50%, the pulsating heat pipe oscillates better; the pulsation frequency is relatively high; and the temperature difference between the end of the cold and hot sections is small, the temperature difference is between 3 and 7 °C, and the performance of heat transfer is better. However, when the liquid charging rate is 30% and the heat source is 70 °C, the thermal resistance is increased to 0.016 K/W, and the equivalent thermal conductivity is reduced. When the performance of heat transfer is changed to 0.016 K/W and the equivalent thermal conductivity is reduced, the coefficient decreases, and the heat-transfer performance becomes weaker.