This study reports a novel strategy by using polyaniline nanofibers (PANFs) to modify membrane-electrode assembly (MEA) for improving direct methanol fuel cell (DMFC) performance. First of all, a series of PANFs emeraldine salt was synthesized and characterized. Then, we investigated the effect of PANFs layout in MEA on DMFC performance. Three different placements to incorporate the as-synthesized PANFs in anodes include (1) placing a layer of PANFs between catalyst layer (CL) and proton exchange membrane (PEM), (2) mixing with catalyst slurry and coating onto gas diffusion layer (GDL), and (3) placing a layer of PANFs between CL and GDL. Polarization curves indicate that the third method is superior to the others and is adopted as the incorporation layout thereafter. Both methanol transport resistance and methanol crossover of the PANFs-modified MEA are studied further. The DMFC incorporated with H2SO4-doped PANFs obtained after the re-doping process with 2 mol L-1 H2SO4 performs a power density as high as 53 mW cm(-2), about 20% higher than that of the pristine one without PANFs incorporation. However, an excessive doping level may result in a higher methanol transport resistance due to PANFs aggregation and thus deteriorate DMFC performance. This study provides a simple and effective way by placing a layer of PANFs between CL and GDL in anode to act as methanol transport regulator and improve DMFC performance consequently. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.