D-arginine oligomers have been widely used as intracellular delivery vectors both in in vitro and in vivo application. Nevertheless, their internalization pathway is obscure and conflicting results have been obtained concerning their intracellular distribution. In this study, we demonstrate that octa-D-arginine (r8) undergoes diffuse localization throughout the cytoplasm and nucleus even at low concentrations and that r8 (r: D-arginine) enters the cells via direct membrane translocation, unlike R8 (R: L-arginine), of which endocytosis is the major internalization pathway. The observation that R8 and r8 enter the cells through two clearly distinct internalization pathways suggests that the backbone stereochemistry affects the uptake mechanism of oligoarginines.