Voltage‐gated sodium channels hold an important role in nerve-transmission signalling leading to the sensation of different kinds of pain. Na_V1.7 subtype is a potential target for pain treatment as it is primarily expressed in peripheral sensory nerves. Conotoxins are well-known for their analgesic activity. Among them, KIIIA is the smallest and most studied μ‐conotoxin that targets Na_V1.7. KIIIA has three disulfide bonds. It has been proven that KIIIA forms two non-native isomers under oxidative conditions. However, there no study to date has focused on the native KIIIA. Hereby, in our study, we chemically synthesized the three μ‐conotoxin KIIIA isomers, which contain different disulfide connectivity. Fmoc solid phase peptide synthesis and a number of cysteine protecting groups were utilized to approach the desire KIIIA isomers. The safety-catch cysteine protecting group 4,4'-dimethylsulfinylbenzhydryl was found to be the most efficient protecting group for directing disulfide connectivity of μ-KIIIA. The three isomers were distinguishable via NMR structure analysis and displayed different bioactivity/selectivity upon examination across Na_V subtypes. This study highlights the importance of activity investigation of different disulfide isomers as peptides displaying native connectivity may not always be the most active or have the most desired subtype selectivity profile.