We successfully characterized the effect of methanol treatment on the nanoscopic structures of a nafion film which is widely used in direct methanol fuel cells (DMFCs). Atomic force microscopy (AFM) was used to repetitively image a particular region of a nafion sample before and after methanol solutions were dropped onto the nafion film and dried in air. When the surface was treated with 20% methanol for 5 min, many nanopores appeared on the surface. The number of nanopores increased when the sample was treated twice or thrice. By repetitive AFM imaging of a particular region of the same sample, we found that the shapes of the nanopores were deformed by the repeated methanol treatment, although the size of the nanopores had not significantly changed. The creation of the nanopores was affected by the concentration of methanol. Our results directly visualized the effects of methanol treatment on the surface structures of a nafion film at nanoscale levels for the first time.