Most of scorpion toxins affecting voltage-gated K+ channels (KTxs) contain a functional dyad composed of a lysine and an aromatic amino acid separated by a suitable distance. By means of two-electrode voltage clamp technique, we describe functional characterization of two Mesobuthus martensii KTxs (BmP02 and BmP03) without the dyad. These two toxins differ by only one single residue at site 16 (K16N) but they display differential affinities on insect and mammalian Shaker-type K+ channels expressed in Xenopus oocytes. At 1 μM concentration, BmP02 and BmP03 inhibited currents of rKv1.1, rKv1.2, rKv1.3, and Shaker IR, but lacked detectable activity on rKv1.4. The half-inhibitory concentrations (IC50) of BmP02 for rKv1.1, rKv1.2, rKv1.3 and Shaker IR channels are 1.95 μM, 4.40 μM, 7 nM and 20.44 μM, respectively. For BmP03, the corresponding IC50 values for these channels are 5.48 μM, 530 nM, 85.4 nM, and 4.64 μM, respectively. Affinity variation (more than 10-fold) between BmP02 and BmP03 on rKv1.3 indicates functional importance of a cationic side chain at site 16. A pH-dependent experiment and a double mutant cycle analysis suggest that the residue K16 resides on the channel-facing surface of the toxin and within 5 Å of rKv1.3 position 401. These two toxins block rKv1.3 in a weak voltage-dependent manner and both slightly shift the current activation curve to positive potentials. Our work is thus crucial to further understanding structure–function relationship of KTxs without a functional dyad.