A synthetic amphipathic α-helical model peptide, KLW, displays non-cell selective cytotoxicity. To investigate the effects of l- or d-Pro kink incorporation into hydrophobic or hydrophilic helix face of KLW on structure, cell selectivity, and membrane-binding affinity, we designed a series of four peptides, in which Leu 9 and Lys 11 in the hydrophobic and hydrophilic helix face of KLW, respectively, are substituted with l- or d-Pro. A l- or d-Pro substitution (KLW-L9P or KLW-L9p) of Leu 9 at the hydrophobic helix face of KLW induced a more significant reduction in hemolytic activity with improved antibacterial activity than that (KLW-K11P or KLW-K11p) of Lys 11 in the hydrophilic helix face. In addition, d-Pro-containing peptides (KLW-L9p and KLW-K11p) displayed less hemolytic activity than l-Pro-containing peptides (KLW-L9P and KLW-K11P). Tryptophan fluorescence studies revealed that bacterial cell selectivity of KLW-L9P, KLW-L9p, and KLW-K11p is closely related to selective interactions with negatively charged phospholipids. CD analysis revealed that l- or d-Pro incorporation into KLW reduces the α-helicity of the peptide and d-Pro incorporation induces more significant disruption in α-helical structure than l-Pro incorporation. Our results collectively suggest that d-Pro incorporation into the hydrophobic helix face of non-cell selective amphipathic α-helical peptides may be useful for the design of novel antimicrobial peptides possessing high bacterial cell selectivity without hemolytic activity.