Scorpion toxins are invaluable therapeutic leads and pharmacological tools which influence the voltage-gated sodium channels. However, the details were still unclear about the structure–function relationship of scorpion toxins on VGSC subtypes. In the previous study, we reported one α-type scorpion toxin Bmk AGP-SYPU1 and its two mutants (Y5F and Y42F) which had been demonstrated to ease pain in mice acetic acid writhing test. However, the function of Bmk AGP-SYPU1 on VGSCs is still unknown. In this study, we examined the effects of BmK AGP-SYPU1 and its two mutants (Y5F and Y42F) on hNav1.4 and hNav1.5 heterologously expressed CHO cell lines by using Na+-specialized fluorescent dye and whole-cell patch clamp. The data showed that BmK AGP-SYPU1 displayed as an activator of hNav1.4 and hNav1.5, which might indeed contribute to its biotoxicity to muscular and cardiac system and exhibited the functional properties of both the α-type and β-type scorpion toxin. Notably, Y5F mutant exhibited lower activatory effects on hNav1.4 and hNav1.5 compared with BmK AGP-SYPU1. Y42F was an enhanced activator and confirmed that the conserved Tyr42 was the key amino acid involved in bioactivity or biotoxicity. These data provided a deep insight into the structure–function relationship of BmK AGP-SYPU1, which may be the guidance for engineering α-toxin with high selectivity on VGSC subtypes.