Farzana et al have published the paper entitled with ” Effect of membrane potential on entry of lactoferricin B-derived 6-residue antimicrobial peptide into single Escherichia coli cells and lipid vesicles” (J. Bacteriology, 203, e00021-21, 2021) [Abstract]. It is considered that a short antimicrobial peptide (AMP) (RRWQWR), derived from lactoferricin B (LfcinB), [LfcinB(4-9)], is a cell-penetrating peptide (CPP)-type AMP (Biochemistry, 56, 4419, 2017). In this paper, we found that at low peptide concentrations, a fluorescent probe-labeled LfcinB(4-9) [i.e., Rh-LfcinB(4-9)] entered the cytoplasm of E. coli cells and their spheroplasts without pore formation in their cell membranes, but a H+ ionophore suppressed this entry. We also observed that Rh-LfcinB(4-9) entered the lumen of giant unilamellar vesicles (GUVs) comprising E. coli polar lipid extract (i.e., E. coli lipid-GUVs) without pore formation and the rate of its entry increased with an increase in negative membrane potential of the GUV membrane. These data directly indicate that negative membrane potential enhances the entry of this peptide into E. coli cells, their spheroplasts, E. coli lipid-GUVs, and thus, its bactericidal activity.