Conus venoms have proven a valuable source of disulfide-rich peptides with the ability to block voltage-gated sodium channel (NaV1) subtypes.  Of particular interest are inhibitors of  NaV1.3 as pharmacological tools for the study/treatment of neuropathic pain associated with spinal cord injury.  The recently described µ-conopeptide from Conus bullatus (µ-BuIIIB) was shown to block  NaV1.3 with low micromolar potency (Kd = 0.2 µM) making it the most potent peptidic inhibitor of this subtype described to date.  Unfortunately, this peptide also blocked NaV1.2 (neuronal) and NaV1.4 (skeletal muscle) subtypes.  Furthermore, oxidative folding of µ-BuIIIB results in numerous folding isoforms, making it difficult to obtain sufficient quantities of the active form of the peptide for detailed SAR studies.  We report the design, synthesis, and testing µ-BuIIIB analogues possessing a disulfide-deficient, diselenide-containing scaffold expected to simplify the synthetic route and facilitate rapid identification of important amino acid residues for NaV1.3 block.  Our results suggested that, similar to other µ-conopeptides, C-terminal residues were most crucial for NaV1 block (Trp-16, Arg-18, and His-20).  Importantly, Ala-replacement of Glu-3 actually resulted in an analogue with increased potency for NaV1.3 (Kd = 0.07 µM), implicating this position as a potential site for modification for increased potency and/or selectivity.  Further examination of this position revealed that increased negative charge through γ-carboxyglutamate (Gla) replacement decreased potency (Kd = 0.33 µM) while positively-charged residues (Dab) actually improved block.  These results are expected to lead to the design and synthesis of BuIIIB-based analogues with increased potency and/or selectivity for NaV1.3.