Background: Anticancer Peptides (ACPs) have received increasing attention as a promising class of novel anticancer agents owing to their potent and rapid cytotoxic properties. In this study, we aim to investigate the effects of cationicity and hydrophobicity in modulating the cytotoxicity of PtxC, a class of ACP from the leafy mistletoe Phoradendron tomentosum against the MDA-MB-231 and Vero cells.Methods: We designed a series of four PtxC analogues (PA1 – PA4) by residual substitutions with specific amino acids to introduce the specific charge and hydrophobicity alterations to the analogues. The cytotoxicity strength of the PtxC analogues on MDA-MB-231 and Vero cells was tested using MTT assay 24 hours post-treatment. Results: PA1, PA2, and PA4 displayed marked increases in cytotoxicity against both MDA-MB- 231 and Vero cells and can be ranked in the order of PA2 > PA4 > PA1 > PtxC > PA3. Sequence- activity relationship analyses of the designed analogues showed that an increase in the level of cationicity and hydrophobicity correlated well with the enhanced cytotoxic activity of PtxC analogues. This was observed with PA1 (netC +8) and PA2 (netC +10) in comparison to PtxC (netC +7). A similar finding was observed for PA4 (GRAVY +0.070) in contrast to PtxC (GRAVY -0.339). Three-dimensional modelling predicted a double α-helix structure in PtxC class of ACP. The larger first helix in PA2 and PA4 was suggested to be responsible for the enhanced cytotoxicity observed. Conclusion: The critical role of cationicity and hydrophobicity in enhancing cytotoxicity of PtxC class of ACPs was clearly demonstrated in our study. The current findings could be extrapolated to benefit peptide design strategy in other classes of ACPs toward the discovery of highly potent ACPs against cancer cells as potential novel therapeutic agents.