Novel proton exchange membranes have been prepared by in synthesis functionalization of a polyurethane matrix with a sulfonic group containing chain terminals. The synthesis procedure was based on the use of two polyethylene glycols with nominal molecular weight of 300 and 1 k and 4,4′ dicyclohexylmethane diisocyanate in presence of the sodium salt of isethionic acid as a donor of the sulfonic group. Glycerol was added in order to improve by reticulation the stability of the cast films. The membranes were characterized in terms of swelling, morphology, methanol permeability, proton conductivity, and ion exchange capacity. The best H2 /air cell performance was achieved at 80 °C with a maximum power density of 16.9 mW/cm2 at a voltage of about 0.35 V. Polyurethane based ionomeric membranes have proved to be interesting candidates for proton exchange membrane fuel cells (PEMFC) applications.