μ-Conotoxin GIIIA, a peptide toxin isolated from Conus geographus, preferentially blocks the skeletal muscle sodium channel Na_V1.4. GIIIA folds compactly to a pyramidal structure stabilized by three disulfide bonds. To assess the contributions of individual disulfide bonds of GIIIA to the blockade of Na_V1.4, seven disulfide-deficient analogues were prepared and characterized, each with one, two, or three pairs of disulfide-bonded Cys residues replaced with Ala. The inhibitory potency of the analogues against Na_V1.4 was assayed by whole cell patch-clamp on rNa_V1.4, heterologously expressed in HEK293 cells. The corresponding IC₅₀ values were 0.069 ± 0.005 μM for GIIIA, 2.1 ± 0.3 μM for GIIIA-1, 3.3 ± 0.2 μM for GIIIA-2, and 15.8 ± 0.8 μM for GIIIA-3 (-1, -2 and -3 represent the removal of disulfide bridges Cys3–Cys15, Cys4–Cys20 and Cys10–Cys21, respectively). Other analogues were not active enough for IC₅₀ measurement. Our results indicate that all three disulfide bonds of GIIIA are required to produce effective inhibition of Na_V1.4, and the removal of any one significantly lowers its sodium channel binding affinity. Cys10–Cys21 is the most important for the Na_V1.4 potency.