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Raising the operating temperature is considered to be an effective method to improve the output performance of proton exchange membrane fuel cells (PEMFCs). In this paper, the effects of inlet relative humidity in the anode (RHa) and cathode (RHc) on the polarization curve and impedance spectra of a single rotating serpentine PEMFC were investigated by experimental method at the operating temperature of 90 °C. It was found that the output performance is the smallest in the high RH case (RHa90%/RHc90%) due to the largest mass transfer resistance. However, the ohmic resistance in the dry case (RHa50%/RHc50%) is the highest, and it shows better output performance at more than 1.0 A/cm² because of the lowest mass transfer resistance. The impact of the changes in the RHa value on the polarization curve is more apparent than that of the RHc changes at high current density. The largest power density can be attained and the efficiency can reach 24.4% when the RHa is 90% and RHc is 50%.