A passive check valve without a subsidiary channel that effectively increases the thermal performance of pulsating heat pipes (PHPs) is proposed. To realize this, topology optimization is employed to maximize the diodicity (Di) of the check valve. The proposed passive check valve consists of a pair of hook-shaped structures and has a Di value of 2.2. PHPs with overall dimensions of 53.5×80×1.5 mm3 are fabricated to investigate the effects of the passive check valve on the thermal performance and the flow behaviors of PHPs. A ten-turn closed-loop serpentine square channel with a height and an average width of 0.6 mm and 2 mm is engraved on a silicon plate and then covered with a glass plate for flow visualization. A total of twenty passive check valves are implemented into the entire channel at the locations where the condenser section and the adiabatic section meet. HFE-7000 is used as the working fluid and the filling ratio is fixed at 48% by volume. The experimental results on PHPs with the single-diameter channel show that the proposed passive check valves significantly improve the thermal performance of PHPs: 48% and 81% reduction in the thermal resistance in the vertical orientation and the horizontal orientation, respectively. Also, the experimental results on PHPs with the dual-diameter channel show that the passive check valves effectively improve the thermal performance over the wide operating range regardless of the orientation of PHPs: 33% and 34% reduction in the thermal resistance in the vertical orientation and the horizontal orientation, respectively. It is found through high-speed photography that this enhanced performance occurs because the passive check valves significantly increase the time-averaged volumetric fraction of vapor in the condenser section by 5 times from 0.09 to 0.45. This stems from the fact that the passive check valves effectively make the working fluid flow 3.5 times more preferentially in one direction over the other.