Uperin 3.5 is a remarkable natural peptide obtained from the skin of toadlets comprised of 17 amino acids which exhibits both antimicrobial and amyloidogenic properties. Molecular dynamics simulations were performed to study the β-aggregation process of uperin 3.5 as well as two of its mutants, in which the positively charged residues Arg7 and Lys8 have been replaced by alanine. All three peptides rapidly underwent spontaneous aggregation and conformational transition from random coils to beta-rich structures. The simulations reveal that the initial and essential step of the aggregation process involves peptide dimerization and the formation of small beta-sheets. A decrease in positive charge and an increase in the number of hydrophobic residues in the mutant peptides lead to an increase in the rate of their aggregation.