Scorpion toxins are invaluable source of therapeutic leads and pharmacological tools which produce influence on the voltage gated sodium channels. In the previous study, our group has reported BmK AGP-SYPU1 (64 amino acids), one scorpion toxin with both potential α-type and β-type scorpion characteristics and analgesic activity in vivo, act as an activator to hNav1.4 and hNav1.5. Additionally, conserved aromatic amino acids Tyr5 and Tyr42 played important roles in bioactivity of BmK AGP-SYPU1 on hNav1.4 and hNav1.5. However, the anti-nonceptitor mechanisms of BmK AGP-SYPU1 referred in vivo have not been clarified yet. The roles of Tyr5 and Tyr42 have still kept unclear in the interaction of BmK AGP-SYPU1 with other VGSCs. In this study, in order to give a deep insight into the relationship of structure and function, the effects of BmK AGP-SYPU1 and its two mutants (Y5F and Y42F) on hNav1.7, which has emerged as a key molecule involved in pain processing, were examined by using Na+-specialized fluorescent dye and the whole-cell patch clamp. The data showed that BmK AGP-SYPU1 also displayed as an activator to hNav1.7 with the potential characteristic of α-type and β-type scorpion toxin. Both Tyr5 and Tyr42 were the key amino acids to the functional properties of BmK AGP-SYPU1 when interacting with hNav1.7, and the Tyr42 was involved in the preferences of the toxin to distinct action sites of hNav1.7. On the whole, these data provided more information about the structure of BmK AGP-SYPU1 related to its function upon hNav1.7, and may bring some clues for engineering scorpion toxins to achieve high bioactivity with lower side effects.