Cyclotides, a large family of peptides from plants, are exceptionally stable and characterized by a cyclic structure and three disulfide-bonds arranged in a cystine knot. Most likely evolved to protect plants against pests and herbivores, cyclotides also exhibit anticancer, anti-HIV and pesticidal activities. Recently we have found that cyclotides can be internalized into human cells at non-toxic concentrations. Their exceptional stability, bioactivities and cell-penetrating properties make cyclotides exciting templates in drug design to inhibit intracellular targets. To assist in the design of novel drugs based on cyclotides, their cell-penetrating properties were examined using biophysical methodologies with model membranes and intact cells. Lipid selectivity and cell-penetration mechanism of the prototypic cyclotide, kalata B1, and analogues were studied by fluorescence spectroscopy, surface plasmon resonance, flow cytometry and confocal microscopy. The cell-penetration efficiency of kalata B1 correlated with its ability to target phosphatidylethanolamine-phospholipids and insert into lipid membranes and its mode-of-action can be summarized in four steps: 1) targeting of PE-phospholipids through specific interactions; 2) insertion into the lipid membrane; 3) local membrane disturbances and 4) cell-penetration. Knowledge on the membrane specificity of cyclotides may help in improving their intrinsic bioactivities and cell-penetrating properties, decreasing toxicity and assisting in the design of novel drugs.