In this study, a novel membrane (Nafion HP JP) was studied and investigated intensively for use as a proton exchange membrane for fuel cell. A standard membrane, Nafion NRE212, was also studied under the same conditions for comparison. Fourier transform infrared spectroscopy, thermogravimetric analysis, wide-angle x-ray diffraction, atomic force microscope (AFM), proton conductivity, and mechanical test were used to study the structure and properties of the membranes. In addition, methanol permeability was measured as a function of methanol concentration. Nafion HP JP membrane exhibited a lower methanol permeability over the concentration range studied. Furthermore, Nafion HP JP has Young’s modulus of 88.3 MPa and tensile strength of 13.2 MPa which are higher than those of Nafion NRE212 (39.85 and 9.5 MPa, respectively). AFM data confirmed the higher value of water uptake for Nafion HP JP membrane. Free volume distribution data measured using positron annihilation lifetime (PAL) showed that the novel membrane has a smaller free volume hole size than that of Nafion NRE212. Moreover, the cell performance of the single cell test using the investigated membrane, hydrogen as fuel and oxygen as oxidant, was investigated at different temperatures and humidities. The power density delivered using Nafion HP JP is higher than that from Nafion NRE212 especially at 50 °C and 30% relative humidity. In addition, the membrane durability of both samples was performed for about 220 h and Nafion HP JP has an open circuit voltage reduction rate of 0.136 mV/h that nearly half that for Nafion NRE212 membrane. The optimal concentration for direct methanol fuel cell DMFC operation using both membranes was found to be 2 M which totally agrees with methanol permeability and PAL data. The obtained results reflect that Nafion HP JP shows great potential for fuel cell applications.