BmK I, a site-3-specific modulator of VGSCs (voltage-gated sodium channels) from the Chinese scorpion Buthus martensi Karsch, can induce spontaneous nociception and hyperalgesia and generate epileptiform responses in rats, which is attributed to the modulation of VGSCs in the neural system. However, which VGSC subtype is targeted by BmK I remains to be identified. Using two-electrode voltage-clamp recording, we studied the efficacy and selectivity of BmK I to three neuronal VGSCs co-expressed with the auxiliary β1 subunit in Xenopus oocytes. Results revealed that BmK I induced a large increase in both transient and persistent currents in mNav1.6α/β1 (where m indicates mouse), which correlated with a prominent reduction in the fast component of inactivating current. In comparison, BmK I-increased currents of rNav1.2α/β1 (where r indicates rat) and rNav1.3α/β1 were much smaller. The EC50 values of BmK I for rNav1.2α/β1 (252±60 nM) and mNav1.6α/β1 (214±30 nM) were similar and roughly half of that for rNav1.3α/β1 (565±16 nM). Moreover, BmK I only accelerated the slow inactivation development and delay recovery of mNav1.6α/β1 through binding to the channel in the open state. Residue-swap analysis verified that an acidic residue (e.g. Asp1602 in mNav1.6) within the domain IV S3-S4 extracellular loop of VGSCs was crucial for the selectivity and modulation pattern of BmK I. Our findings thus provide the molecular determinant explaining the divergent and intriguing behaviour of neuronal VGSCs in response to site-3-specific modulators, indicating that these subtypes play different roles in BmK I-induced hyperexcitablity in rat models.