The conserved tridisulfide array of the α-defensin family imposes a common triple-stranded, β-sheet topology on peptides that may have highly diverse primary structures, resulting in differential outcomes after targeted mutagenesis. In the mouse cryptdin-4 (Crp4) and macaque myeloid RMAD4 α-defensins, complete substitutions of Arg with Lys (R/K) affect bactericidal peptide activity very differently. R/K mutagenesis attenuates Crp4 but RMAD4 activity remains mostly unchanged. Here, we show that the differential biological effect of Lys for Arg replacements can be understood by the distinct phase behavior of the experimental peptide-lipid system. In Crp4, Small Angle X-ray Scattering (SAXS) analyses showed that Arg to Lys replacements shifted the induced nanoporous phases to a different range of lipid compositions compared to the Arg-rich native peptide, consistent with the attenuation of bactericidal activity by R/K mutations. In contrast, such phases generated by RMAD4 were largely unchanged. The concordance between SAXS measurements and biological activity provide evidence that specific types of α-defensin-induced membrane curvature generating tendencies correspond directly to bactericidal activity via membrane destabilization.