The molecular interactions between venomous peptides and potassium channels have extensively enriched the knowledge of diverse peptide pharmacology, and the in-depth understanding of general features of the various peptide functions remains a formidable challenge. In this work, the role of peptide basic residues in peptide pharmacology was first investigated. Although the venomous BmK-NSPK peptide had the critically conserved functional residues occurring in its similar and potent potassium channel-inhibiting peptides, it was a remarkably weak inhibitor of potassium channels due to fewer basic residues. Additionally, 1 μM BmK-NSPK only inhibited 1.2 ± 1.0%, 1.7 ± 0.70%, 2.3 ± 0.49% and 5.4 ± 0.70% of hKv1.1, hKv1.2, hKv1.3 and hKv1.6 channel currents. The introduction of one or two basic residues in BmK-NSPK-I15K, BmK-NSPK-I18K, BmK-NSPK-I26K and BmK-NSPK-I18K/I26K could not improve BmK-NSPK activity. The modifications of more than three basic residues were found to continuously improve BmK-NSPK activity, and the corresponding BmK-NSPK-7K and BmK-NSPK-8K mutants could effectively inhibit hKv1.3 channel with IC