The discovery of new antimicrobials that use novel modes of action and avoid antibiotic resistance is crucial to fight microbial infections. Cationic antimicrobial peptides (AMPs) have been regarded as promising antibiotic candidates and knowledge on their mode of action is important for further improvement. Here we investigate the mechanism of action of Sub3, an AMP with high activity against Gram-negative and Gram-positive bacteria and low cytotoxicity. Using model membranes and bacterial cells with fluorescence spectroscopy, flow cytometry, Zeta potential and atomic force microscopy imaging we found that the action of Sub3 involves both membrane and intracellular targeting. At lethal concentrations Sub3 targets the bacteria membrane, trough electrostatic attractions, and internalizes inside the bacteria without permeabilizing the cell membrane. In addition, Sub3 can also internalize into human cells without being toxic to them. The observation that Sub3 is able to internalize into host cells, is of major relevance because in addition to its potent activity against bacteria, Sub3 has promise for treating locally-infected mammalian cells which are difficult to treat.