Self-assembly property of a new liquid crystalline perylene diimide derivative, N,N'-bis[3-[2-[2-[2-(2-cyanoethoxy)ethoxy]ethoxy]ethoxy]-propyl]perylene-3,4:9,10-tetracarboxyldiimide (PPE4CN), was studied at the air/water interface by epifluorescence microscopy and on mica by environmental scanning electron microscopy (ESEM) and atomic force microscopy (AFM). The self-assembly process started at surface pressure 5 mN/m. Molecular orientation changes of the PPE4CN molecules accompanied the self-assembly process at the air/water interface. It was found that the molecular orientation changed from face-on to edge-on orientation in the self-assembly process. These orientation changes were strongly supported by the analysis of the surface pressure-area isotherms, and by the topographic measurements at micro and nanoscale levels, including ESEM and AFM microscopies.