Cyclotides are cyclic plant proteins with potent cytotoxic effects. Here we systematically probed the importance of surface-exposed charged amino acid residues of the cyclotide cycloviolacin O2, using a strategy involving chemical modifications. We show that the single glutamic acid plays a key role for the cytotoxicity: methylation of this residue produced a 48-fold decrease in potency. Virtually no change in potency was observed when masking the single arginine residue using 1,2-cyclohexanedione, while acetylation of the two lysine residues reduced the potency 3-fold. The derivative with modifications at both arginine and lysine residues showed a 7-fold loss of potency. In addition, we show that the activity is dependent on an intact disulfide network and that the short sequences between the six cysteine residues, that is, the backbone loops, are devoid of cytotoxic activity.