In nature, animals and plants use painful toxins as defensive mechanism against predators or competitors. Most of toxins act as irritants or cause pain by interacting with functional domains of ion channels.A target of these painful toxins is the transient receptor potential vanilloid 1 (TRPV1) which is a cation-selective ion channel expressed in primary sensory neurons. TRPV1 is an pain receptor modulated by multiple noxious stimuli as high temperature (>42°), low pH (< 6) or irritants compounds.The Double Knot toxin (DkTx) from Ornithoctonus huwena spider, is a polypeptide which interacts with the extracellular surface of TRPV1 disturbing the selectivity filter and driving the activation of the channel. Even when interaction surface of TRPV1 and DkTx has been identified by site directed mutagenesis and a cryo-EM structure, the structure of DkTx and its binding mechanism remain unknown. In order to understand the atomic interactions involved in DkTx binding, we build a homology model of DkTx toxin which was subsequently docked to the TRPV1 putative binding surface structure to then performing molecular dynamics simulations. Our results suggest that each domain of DkTx toxin independently interact with a single subunit of the TRPV1 tetramer and these interaction network is strongly stabilized by charged residues in both interacting surfaces.