Tachyplesin I, a peptide isolated from the haemocytes of the Japanese horseshoe crab, is positively-charged, possess two disulfide bonds and a beta-hairpin structure. It belongs to the group of host defence peptides, which often share the ability to associate with the negatively charged cell membrane of microbes and exhibit antimicrobial properties through membrane disruption, pore formation or cell penetration to reach an intracellular target. Similar to microbial cells, the surface of cancer cell membranes is more negatively charged than healthy cells. In this study, we were specifically interested in exploring the anticancer properties of Tachyplesin I and its mode of action. To achieve this, we have designed a series of cyclic analogues of Tachyplesin I, with different hydrophobicity and charge, and have examined their toxicity and selectivity towards different cancer types. In addition, we have conducted studies with model membranes to examine membrane-binding affinity and the ability to disrupt lipid bilayers. With our studies we confirmed that Tachyplesin I has anticancer properties, we identified key residues and designed an analogue that is more potent than the native peptide. The gained information will be used to guide the design of novel analogues and to improve potency and selectivity towards specific types of cancer cells.