The use of nanocarriers decorated with penetration-enhancing agents (PEAs) is considered to be a promising approach for efficient transdermal delivery. In this study, we developed short amphiphilic skin-penetrating peptides (17 amino acids) that functioned not only as PEAs but also as building blocks of nanocarriers without the incorporation of additional macromolecules for self-assembly and guest molecule encapsulation. Interestingly, varying only two amino acids in the hydrophobic moiety of the peptides resulted in significantly different self-assembly behavior, thermal stability, protease resistance, and skin-penetration efficiency in a human skin model. The analysis of the peptide secondary structure revealed that such characteristic changes arose due to the sequence variation-mediated conformational change in the hydrophobic block. These findings hold significant promise for the development of simple and effective delivery systems exhibiting controllable supramolecular properties.