The effects of gravitational force on the orientation-dependent performance of portable proton exchange membrane (PEM) fuel cell using serpentine flow channels were investigated by the measurement and analysis of polarization curves. Whether the removal of produced water in the cathode flow channel is resisted or assisted by the gravity depends on the orientation variation, flow direction, and flow channel distribution of a fuel cell. This gravity will then affect the fuel cell performance, especially for fuel cells operating at a high current density. The results show that a fuel cell with perpendicular flow channel distribution and cathode gas flow in vertical direction requires a longer distance of pushing liquid droplets against gravity to remove the produced water, which is difficult to expel the produced water from the flow channels, and the performance reduction is obviously in high current density. A fuel cell operating in a normal position achieves higher performance than one operating in a horizontal position, except the cathode gas flow in vertical direction and feed from lower inlet. Furthermore, for a fuel cell operating in a horizontal position with anode below the membrane, gravitational force transports the water to the anode and blocks the fuel channel in the gas diffusion layer. This leads a fuel cell operating in high current densities with the cathode below the membrane performs better than one with the cathode above the membrane. Therefore, to reduce the effects of gravity on the orientation-dependent performance, a fuel cell with parallel flow channel distribution and feeding the cathode gas from the upper inlet port is recommended in this study.