A collaborative experimental and theoretical study on the dependence of the infrared (IR) spectrum of hydrated Nafion,electrolyte membrane on the hydration number is investigated in great detail. Experimental time-resolved attenuated total reflection Fourier transform IR spectroscopic results show that Nafion membrane has a unique IR peak intensity dependence on the hydration number. Calculated IR spectra indicate that this unique IR peak intensity dependence is correctly reproduced not for the singly hydrated Nafion but for the doubly hydrated Nafion. This result strongly supports the relay mechanism of the proton conductance, in which protonated water dusters are relayed by the side chains through the doubly hydrated sulfonic acid groups under low humidity conditions.