Human α-defensin 5 (HD5) is a 32-residue cysteine-rich host-defense peptide that exhibits broad-spectrum antimicrobial activity and contributes to innate immunity in the human gut and other organ systems. Despite many years of investigation, its antimicrobial mechanism of action remains unclear. In this work, we report that HD5ox, the oxidized form of this peptide that exhibits three regiospecific disulfide bonds, causes distinct morphological changes to Escherichia coli and other Gram-negative microbes. These morphologies include bleb formation, cellular elongation, and clumping. The blebs are up to ∼1 μm wide and typically form at the site of cell division or cell poles. Studies with E. coli expressing cytoplasmic GFP reveal that HD5ox treatment causes GFP emission to localize in the bleb. To probe the cellular uptake of HD5ox and subsequent localization, we describe the design and characterization of a fluorophore-HD5 conjugate family. By employing these peptides, we demonstrate that fluorophore-HD5ox conjugates harboring the rhodamine and coumarin fluorophores enter the E. coli cytoplasm. On the basis of the fluorescence profiles, each of these fluorophore-HD5ox conjugates localizes to the site of cell division and cell poles. These studies support the notion that HD5ox, at least in part, exerts its antibacterial activity against E. coli and other Gram-negative microbes in the cytoplasm.