Cellulose whiskers (CWs) were explored as effective nanostructures to manipulate the microstructure of Nafion® membranes for high-performance fuel cell applications. Electrochemical impedance measurements revealed that not only the proton conductivity of the nanocomposite membranes comprising 5 wt% CWs was remarkably increased, but the higher conductivity was also retained at higher temperatures (>100 °C). Moreover, it was found that the inclusion of CWs into a Nafion® matrix provides considerably suppressed methanol permeability. The direct methanol–air single fuel cell test using 5 M methanol solution at 70 °C showed a higher maximum power density of 91 mW cm−2 for the nanocomposite membrane, compared to 28 and 47 mW cm−2 for unmodified Nafion®. Hence, CWs could be considered as effective and elegant nanostructures to regulate the ionic microstructure and alleviate critical drawbacks of Nafion membranes for commercial direct methanol fuel cell applications.