Tachyplesin-I (TI) is a host defense peptide from the horseshoe crab Tachypleus tridentatus that has outstanding potential as an anticancer therapeutic lead. Backbone cyclized TI (cTI) has similar anticancer properties to TI but has higher stability and lower hemolytic activity. We designed and synthesized cTI analogues to further improve anticancer potential and investigated structure-activity relationships based on peptide-membrane interactions, cellular uptake, and anticancer activity. The membrane-binding affinity and cytotoxic activity of cTI were found to be highly dependent on peptide hydrophobicity and charge. We describe two analogues with increased selectivity toward melanoma cells and one analogue with the ability to enter cells with high efficacy and low toxicity. Overall, the structure-activity relationship study shows that cTI can be developed as a membrane-active antimelanoma lead, or be employed as a cell penetrating peptide scaffold that can target and enter cells without damaging their integrity.