Proficient transport vectors called cell-penetrating peptides (CPPs) internalize into eukaryotic cells mostly via endocytic pathways and facilitate the uptake of various cargo molecules attached to them. However, some CPPs are able to induce disturbances in the plasma membrane and translocate through it seemingly in an energy-independent manner. For understanding this phenomenon, giant plasma membrane vesides (GPMVs) derived from the cells are a beneficial model system, since GPMVs have a complex membrane composition comparable to the cells yet lack cellular energy dependent mechanisms. We investigated the translocation of arginine-rich CPPs into GPMVs with different membrane compositions. Our results demonstrate that lower cholesterol content favors accumulation of nona-arginine and, additionally, sequestration of cholesterol increases the uptake of the CPPs in vesicles with higher cholesterol packing density. Furthermore, the proteins on the surface of vesicles are essential for the uptake of arginine-rich CPPs: downregulation of nudeolin decreases the accumulation and digestion of proteins on the membrane suppresses translocation even more efficiently.