Recent studies on peptide fibrillogenesis by the de novo method as well as amyloidogenic proteins including prion proteins and Alzheimer’s β-peptides have provided insights into the conformational changes, such as α-helix to β-structure, involved in folding and misfolding processes. We have found that an exposed hydrophobic nucleation domain at N-terminal causes a structural transition of a peptide from α-helix to β-fibril. It became clear that N-terminal acyl groups of particular lengths in a 2α-helix peptide caused the peptide to undergo an α-to-β transition. The peptide with the octanoyl group (C8-2α) showed the highest rate of transformation. The study of the designed peptides revealed that these α-to-β transitions were closely related to the initial α-helix conformation and its stability. Engineering peptides that undergo α-to-β transitions are attractive not only to the study of pathogenic proteins such as prion proteins, but also to the control of self-assembly of peptides, which will lead to the development of peptidyl self-assembling materials.