The so-called Kunitz domain proteins contain three highly conserved disulfide bridges and include well-characterized serine protease inhibitors like the bovine pancreatic trypsin inhibitor. Protease inhibitors show striking structural similarities with certain venom peptides which inhibit voltage-gated K channels (Lancelin et al. 1994, Nat. Struct. Biol. 1:246–250). Conkunitzin-S1 (Conk-S1) from the venom of Conus striatus and its homologue Conk-S2 show a backbone fold similar to that of the canonical Kunitz inhibitors, with only 2 disulfide bonds stabilizing their structure. Conk-S1 is known to inhibit voltage gated Shaker potassium currents and has been shown to selectively block Kv1.7. In rodents Conk-S1 enhances insulin secretion, in a glucose dependent fashion, likely via block of Kv1.7 mediated currents (Finol-Urdaneta et al.). Here we demonstrate that Conk-S1 (at pH 7.6 and 4.2) and Conk-S2 (at pH 4.2) inhibit trypsin activity in a standard colorimetric assay. Conkunitzins are larger than most pore-blocking peptides (Conk-S1: 60, and Conk-S2: 65 amino acids), from which several basic and aromatic residues may potentially interact with cation-binding surfaces. Substitution Y51A in Conk-S1 abolishes protease inhibition and decreases affinity towards human Kv1.7 channels. Voltage-clamp studies confirm Conk-S1 selective targeting of Kv1.7 channels and suggest that such inhibitory mechanism may be based on a combination of direct pore block, and gating modulation. Alanine replacement of either of two basic residues from Conk-S1 results in increased IC50 (K21: wt and pore mutant H341D; or R34: H341D only). Additionally, a gating shift, likely associated with protonation of H341, is inhibited by Conk-S1. Thus, these combined results suggest that inhibition of trypsin and Kv1.7 block are mediated by overlapping parts of Conk-S1.