In this paper, we report the high proton conductivity of a single high-purity Nafion nanofiber (1.5 S/cm), which is an order of magnitude higher than the bulk Nafion film ( approximately 0.1 S/cm). We also observe a nanosize effect, where proton conductivity increases sharply with decreasing fiber diameter. X-ray scattering provides a rationale for these findings, where an oriented ionic morphology was observed in the nanofiber in contrast to the isotropic morphology in the bulk film. This work also demonstrates the successful fabrication of high-purity Nafion nanofibers ( approximately 99.9 wt %) via electrospinning and higher humidity sensitivity for nanofibers compared to the bulk. These results should have a significant impact on fuel cells and sensors.