Negamycin is a natural pseudo-dipeptide antibiotic with promising activity against Gram-negative and Gram-positive bacteria, including Enterobacteriaceae, Pseudomonas aeruginosa, and Staphylococcus aureus, and good efficacy in infection models. It binds to ribosomes with a novel binding mode, stimulating miscoding and inhibiting ribosome translocation. We were particularly interested in studying how the small, positively charged natural product reaches its cytoplasmic target in Escherichia coli. Negamycin crosses the cytoplasmic membrane by multiple routes depending on environmental conditions. In a peptide-free medium, negamycin uses endogenous peptide transporters for active translocation, preferentially the dipeptide permease Dpp. However, in the absence of functional Dpp or in the presence of outcompeting nutrient peptides, negamycin can still enter the cytoplasm. We observed a contribution of the DppA homologs SapA and OppA, as well as of DtpD, a proton-dependent oligopeptide transporter. Calcium strongly improves the activity of negamycin against both Gram-negative and Gram-positive bacteria, especially at concentrations around 2.5 mM, reflecting human blood levels. Calcium forms a complex with negamycin and facilitates its interaction with negatively charged phospholipids in bacterial membranes. Moreover, decreased activity at acidic pH and under anaerobic conditions point to a role of the membrane potential in negamycin uptake. Accordingly, improved activity at alkaline pH could be linked to increased uptake of [3H]negamycin. The diversity of options for membrane translocation is reflected by low resistance rates. The example of negamycin demonstrates that membrane passage of antibiotics can be multi-faceted and that for cytoplasmic anti-Gram-negative drugs, understanding of permeation and target interaction are equally important.