In this paper, we demonstrated that supramolecular structures of amphiphilic tripeptides can be effectively modulated by selecting the appropriate casting solvents. The tripeptide derivatives having an ammonium head and hydrophobic tails were dispersed in three kinds of solvents (nonpolar, medium nonpolar, and polar) followed by casting the solutions onto fleshly cleaved mica. The supramolecular structures in the cast films were observed using an atomic force microscope (AFM). The tripeptide derivative possessing the Ala-Ala-Glu sequence formed needles with nanometer-scale width, micrometer-scale rods, and patterns with molecular-level flatness by casting from H2O, CCl4, and CHCl3, respectively. Since the major driving forces of the assembly are significantly influenced by the polarity of the surrounding media, the assembling process can be altered depending on the nature of the solvents. In polar H2O, the hydrophobic interaction is dominant and the resulting assemblies are supported by hydrogen bonding. ...