Voltage-gated sodium (NaV) channels have been extensively investigated as therapeutic targets for pain. Gympietides are a family of pain-causing peptides derived from Australian stinging trees that modulate NaV1.7 by targeting TMEM233, a transmembrane protein belonging to the dispanin family. This mechanism of action contrasts with that of numerous animal venom-derived neurotoxins, which directly target the pore-forming alpha-subunit of NaV channels to modulate channel activity. In this study we sought to understand the structure-activity relationship between the gympietide Excelsatoxin A (ExTxA) and both TMEM233 and NaV1.7. Protein-protein interactions between peptides and TMEM233 were assessed using interaction assay AlphaScreen. Functional activity at NaV1.7/TMEM233 complex was assessed using FLIPR membrane potential assays and automated patch-clamp electrophysiology. Alanine-scanning mutagenesis and single-residue substitutions identified R3, R13, T22 and D35 as residues critical for activity. ExTxA[R3E] and ExTxA[R13E] lost 6-fold and 12-fold potency at TMEM233 respectively, indicating that they are important for TMEM233 interaction. Conversely, ExTxA[T22I] and ExTxA[D35R] retained comparable binding potencies to TMEM233 as native ExTxA in the AlphaScreen binding assay, yet demonstrated 70% and 80% reductions in efficacy respectively, suggesting that T22 and D35 are primarily involved in NaV1.7 interaction. These findings provide initial insights into the tripartite interaction between gympietides, TMEM233 and Nav1.7, offering a foundation for the rational design of novel dispanin-targeting analgesics.