Cell-penetrating peptides (CPPs) possess the capacity to induce cell entry of themselves and attached molecular cargo, either by endocytosis or by direct translocation. Conformational constraints have been described as one means to increase the activity of CPPs, especially for direct crossing of the plasma membrane. Here, we explored the structure-activity relationship of bicyclic peptides for cell entry. These peptides may be considered minimal analogues of naturally occurring oligocyclic peptide toxins and are a promising scaffold for the design of bioactive molecules. Increasing numbers of arginine residues that are primarily contributing to cell-penetrating activity were introduced either into the cycles, or as stretches outside the cycles, at both ends or at one end only. In addition, we probed for the impact of negatively charged residues on activity for both patterns of arginine substitution. Uptake was investigated in HeLa cells by flow cytometry and confocal microscopy. Overall, uptake efficiency showed a positive correlation with the number of arginine residues. The subcellular distribution was indicative of endocytic uptake. One linear stretch of arginines coupled outside the bicycle was as effective in promoting uptake as substituting the same number of arginines inside the bicycles. However, the internally substituted analogues were more sensitive to the presence of negatively charged residues. For a given bicyclic peptide, uptake was more effective than for the linear counterpart. Introduction of histidine and tryptophans further increased uptake efficiency to comparable levels as that of nonaarginine despite the larger size of the bicyclic backbone. The results demonstrate that both arginine clustering and spatial constraints are uptake-promoting structural principles, an observation that gives freedom in the introduction of cell-penetrating capacity to structurally constrained scaffolds.