Cone snail toxins or conotoxins are often small cysteine-rich peptides which have shown to be highly selective ligands for a wide range of ion channels such as voltage-gated sodium channels (NaVs). NaVs participate in a wide range of electrophysiological processes. Consequently, their malfunction has been associated with numerous diseases. The development of subtype-selective modulators of NaVs remains highly important in the treatment of such disorders. In order to expand our knowledge in the search for novel therapeutics to treat NaV-related diseases, we explored the field of peptide engineering. In the current study, the impact of well considered point mutations into a bioactive peptide that was found to be a very potent and selective inhibitor of NaVs (i.e. Midi R2) was examined. We designed two peptides, named Midi R2[A7G] and Midi R2[H10A, R12A] which have mutations at position 7, and both 10 and 12, respectively. Electrophysiological recordings indicated that an Ala to Gly mutation at position 7 increased IC50-values from the nanomolar range to the micromolar range. For Midi R2[H10A, R12A] at a concentration of 10μM, activity is even reduced to 0-10% for all of the tested NaV-channels. Circular dichroism measurements proved that overall structural conformations did not change. These findings suggest that the minimal space between the second and the third intercysteine loop of Midi R2 is the sequence RRWARDHSR and that His at position 10 and Arg at position 12 are crucial amino acids for the potency and specificity of Midi R2. In this way, new insights into the structure-activity relationships of μ-conotoxins were found.