Pseudhymenochirin-1Pb (Ps-1Pb; IKIPSFFRNILKKVGKEAVSLIAGALKQS) and pseudhymenochirin-2Pa (Ps-2Pa; GIFPIFAKLLGKVIKVASSLISKGRTE) are amphibian peptides with broad spectrum antimicrobial activities and cytotoxicity against mammalian cells. In the membrane-mimetic solvent 50% (v/v) trifluoroethanol-H2O, both peptides adopt a well-defined α-helical conformation that extends over almost all the sequence and incorporates a flexible bend. Both peptides significantly (p < 0.05) stimulate the rate of release of insulin from BRIN-BD11 clonal β-cells at concentrations ≥ 0.1 nM but produce loss of integrity of the plasma membrane at concentrations ≥ 1 μM. Increasing cationicity by the substitution Glu(17) → l-Lys in Ps-1Pb and Glu(27) → l-Lys in Ps-2Pa generates analogues with increased cytotoxicity and reduced insulin-releasing potency. In contrast, the analogues [R8r]Ps-1Pb and [K8k,K19k]Ps-2Pa, incorporating d-amino acid residues to destabilize the α-helical domains, retain potent insulin-releasing activity but are nontoxic to BRIN-BD11 cells at concentrations of 3 μM. [R8r]Ps-1Pb produces a significant increase in insulin release rate at 0.3 nM and [K8k,K19k]Ps-2Pa at 0.01 nM. Both analogues show low hemolytic activity (IC50 > 100 μM) but retain broad-spectrum antimicrobial activity and remain cytotoxic to a range of human tumor cell lines, albeit with lower potency than the naturally occurring peptides. These analogues show potential for development into agents for type 2 diabetes therapy.