Intracellular delivery crossing the endomembrane barrier is the “last mile to target” for nano delivery systems carrying biomacromolecules, including genetic medicines. Nevertheless, a mass of nanomedicines is currently restricted by their equivocal safety and delivery efficiency. Here, we trimmed nano delivery at the cell perspective and established a general strategy independent of nanomaterials. Such a policy broadly facilitates the intracellular delivery of all kinds of tested nanomedicines, subtly by inducing ARF6 GTPases to their overactivated GTP-bound state. We discovered that ARF6, one member of ARF subfamily in small GTPases, regulated intracellular vesicle transport and lipid metabolism through GTP/GDP conversion. More importantly, we demonstrated that ARF6 biased to GTP-bound form induced GTPase overactivation, promoting endocytosis and reducing exocytosis of cargoes, including transferrin proteins and eleven types of nanogranules. This universal effect was mechanistically derived from forming a particular category of hybrid endosomes triggered by overactivated ARF6 via regulating cholesterol-associated vesicles and lipid raft/caveolae pathways. Cargoes were steadily and slowly delivered to the cytoplasm due to the mild microenvironment in hybrid endosomes. Based on these findings, we identified that QS11, a safe small molecule inhibitor of ARF GTPase-activating proteins, enhanced the antitumor efficacy of siEGFR-loaded nanoparticles by inducing ARF6 overactivation. In sum, it demonstrates that the tactics of tuning ARF6 GTPases to GTP-bound form will widely benefit cellular nano delivery.