Abstract Background For a large number of conopeptides basic knowledge related to structure-activity relationships is unavailable although such information is indispensable with respect to drug development and their use as drug leads. Methods A combined experimental and theoretical approach employing electrophysiology and molecular modeling was applied for identifying the conopeptide δ-EVIA binding site at voltage-gated Na+ channels and to gain insight into the toxin's mode of action. Results Conopeptide δ-EVIA was synthesized and its structure was re-determined by NMR spectroscopy for molecular docking studies. Molecular docking and molecular dynamics simulation studies were performed involving the domain IV voltage sensor in a resting conformation and part of the domain I S5 transmembrane segment. Molecular modeling was stimulated by functional studies, which demonstrated the importance of domains I and IV of the neuronal NaV1.7 channel for toxin action. Conclusions δ-EVIA shares its binding epitope with other voltage-sensor toxins, such as the conotoxin δ-SVIE and various scorpion α-toxins. In contrast to previous in silico toxin binding studies, we present here in silico binding studies of a voltage-sensor toxin including the entire toxin binding site comprising the resting domain IV voltage sensor and S5 of domain I. General significance The prototypical voltage-sensor toxin δ-EVIA is suited for the elucidation of its binding epitope; in-depth analysis of its interaction with the channel target yields information on the mode of action and might also help to unravel the mechanism of voltage-dependent channel gating and coupling of activation and inactivation.