The function and properties of peptide-based materials depend not only on the amino acid sequence but also on the molecular conformations. In this paper, we chose a series of peptides G(m)(XXKK)(n)X-NH2 (m = 0, 3; n = 2, 3; X = I, L, and V) as the model molecules and studied the conformation regulation through N-terminus lipidation and their formulation with surfactants. The structural and morphological transition of peptide self-assemblies have also been investigated via transmission electron microscopy, atomic force microscopy, circular dichroism spectroscopy, and small-angle neutron scattering. With the terminal alkylation, the molecular conformation changed from random coil to beta-sheet or alpha-helix. The antimicrobial activities of alkylated peptide were different. C-16-G(3)(IIKK)(3)I-NH2 showed antimicrobial activity against Streptococcus mutans, while C-16-(IIKK)(2)I-NH2 and C-16-G(3)(IIKK)(2)I-NH2 did not kill the bacteria. The surfactant sodium dodecyl sulfonate could rapidly induce the self-assemblies of alkylated peptides (C-16-(IIKK)(2)I-NH2, C-16-G(3)(IIKK)(2)I-NH2, C-16-G(3)(VVKK)(2)V-NH2) from nanofibers to micelles, along with the conformation changing from beta-sheet to alpha-helix. The cationic surfactant hexadecyl trimethyl ammonium bromide made the lipopeptide nanofibers thinner, and nonionic surfactant polyoxyethylene (23) lauryl ether (C12EO23) induced the nanofibers much more intensively. Both the activity and the conformation of the alpha-helical peptide could be modulated by lipidation. Then, the self-assembled morphologies of alkylated peptides could also be further regulated with surfactants through hydrophobic, electrostatic, and hydrogen-bonding interactions. These results provided useful strategies to regulate the molecular conformations in peptide-based material functionalization.