Scorpion toxins affecting K+ channels (KTxs) represent important pharmacological tools and potential drug candidates. Here, we report molecular characterization of seven new KTxs in the scorpion Mesobuthus eupeus by cDNA cloning combined with biochemical approaches. Comparative modeling supports that all these KTxs share a conserved cysteine-stabilized alpha-helix/beta-sheet structural motif despite the differences in protein sequence and size. We investigated functional diversification of two orthologous alpha-KTxs (MeuTXK alpha 1 from M. eupeus and BmP01 from Mesobuthus martensii) by comparing their K+ channel-blocking activities. Pharmacologically, MeuTXK alpha 1 selectively blocked Kv1.3 channel with nanomolar affinity (IC50, 2.36-0.9 nM), whereas only 35% of Kv1.1 currents were inhibited at 3 mu M concentration, showing more than 1271-fold selectivity for Kv1.3 over Kv1.1. This peptide displayed a weak effect on Drosophila Shaker channel and no activity on Kv1.2, Kv1.4, Kv1.5, Kv1.6, and human ether-a-go-go-related gene (hERG) K+ channels. Although BmB01 and MeuTXK alpha 1 have a similar channel spectrum, their affinity and selectivity for these channels largely varies. In comparison with MeuTXK alpha 1, BmP01 only exhibits a submicromolar affinity (IC50, 133.72 +/- 10.98 nM) for Kv1.3, showing 57-fold less activity than MeuTXK alpha 1. Moreover, it lacks the ability to distinguish between Kv1.1 and Kv1.3. We also found that MeuTXK alpha 1 inhibited the proliferation of activated T cells induced by phorbol myristate acetate and ionomycin at micromolar concentrations. Our results demonstrate that accelerated evolution drives affinity variations of orthologous alpha-KTxs on Kv channels and indicate that MeuTXK alpha 1 is a promising candidate to develop an immune modulation agent for human autoimmune diseases. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.002832, 1-11, 2011.